Products and compositions

ABSTRACT

The present invention relates to products and compositions and their uses. In particular the invention relates to nucleic acid products that interfere with target gene expression or inhibit target gene expression and therapeutic uses of such products.

The present invention relates to products and compositions and theiruses. In particular the invention relates to nucleic acid products thatinterfere with target gene expression or inhibit target gene expressionand therapeutic uses of such products.

BACKGROUND

Double-stranded RNA (dsRNA) able to complementarily bind expressed mRNAhas been shown to be able to block gene expression (Fire et al, 1998 andElbashir et al, 2001) by a mechanism that has been termed RNAinterference (RNAi). Short dsRNAs direct gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and have become a useful tool for studying gene function. RNAi ismediated by the RNA-induced silencing complex (RISC), asequence-specific, multi-component nuclease that destroys messenger RNAshomologous to the silencing trigger loaded into the RISC complex.Interfering RNA (iRNA) such as siRNAs, antisense RNA, and micro-RNA areoligonucleotides that prevent the formation of proteins bygene-silencing i.e. inhibiting gene translation of the protein throughdegradation of mRNA molecules. Gene-silencing agents are becomingincreasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226,p 365-379) there are algorithms that can be used to design nucleic acidsbut none is perfect. It may take various experimental methods toidentify potent siRNAs, as algorithms do not take into account factorssuch as tertiary structure or the involvement of RNA binding proteins.Therefore the discovery of a potent nucleic acid with minimal off-targeteffects is a complex process. For the pharmaceutical development ofthese highly charged molecules it is necessary that they can besynthesised economically, distributed to target tissues, enter cells andfunction within acceptable limits of toxicity.

However, delivery of nucleic acids, such as RNA, to cells avoidingdegradation by cellular nucleases, whilst maintaining efficacy andtarget specificity has proved challenging to those in the field ofdeveloping nucleic acid molecules for therapeutic use.

Thus, means for efficient delivery of oligonucleotides, in particulardouble stranded siRNAs, to cells in vivo is becoming increasinglyimportant and requires specific targeting and substantial protectionfrom the extracellular environment, particularly serum proteins. Onemethod of achieving specific targeting is to conjugate a targetingmoiety to the iRNA duplex agent. The targeting moiety helps in targetingthe iRNA duplex agent to the required target site and there is a need todesign appropriate targeting moieties for the desired receptor sites forthe conjugated molecules to be taken up by the cells such as byendocytosis.

However, targeting ligands developed so far do not always translate toin vivo settings and there is a clear need for more efficacious receptorspecific ligand conjugated iRNA duplex agents and methods for theirpreparation for the in vivo delivery of oligonucleotide therapeutics,nucleic acids and double stranded siRNAs.

Rather than a lipid delivery system alone, the present inventionaddresses the structure of the nucleic acid itself. It has beenunexpectedly found that a nucleic acid in accordance with the presentinvention has increased stability, which prevents degradation of thenucleic acid before entry into a cell.

SUMMARY OF INVENTION

A first aspect of the invention relates to a nucleic acid for inhibitingexpression of a target gene in a cell, comprising at least one duplexregion that comprises at least a portion of a first strand and at leasta portion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of RNA transcribed from said targetgene to be inhibited and wherein the terminal nucleotide at the 3′ endof at least one of the first strand and the second strand is an invertednucleotide and is attached to the adjacent nucleotide via the 3′ carbonof the terminal nucleotide and the 3′ carbon of the adjacent nucleotideand/or the terminal nucleotide at the 5′ end of at least one of thefirst strand and the second strand is an inverted nucleotide and isattached to the adjacent nucleotide via the 5′ carbon of the terminalnucleotide and the 5′ carbon of the adjacent nucleotide.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be attached to the adjacent nucleotide via a phosphate group by wayof a phosphodiester linkage.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be attached to the adjacent nucleotide via a phosphorothioate group.The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be attached to the adjacent nucleotide via a phosphorodithioategroup.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be an A or a G.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay form an overhang. The 3′ and/or 5′ inverted nucleotide of the firstand/or second strand may form a blunt end. The first strand and secondstrand of the nucleic acid may be separate strands. The nucleic acid maycomprise a single strand that comprises the first strand and the secondstrand.

The first strand and/or said second strand may be each from 17-35nucleotides in length and the at least one duplex region may consist of19-25 nucleotide base pairs.

The nucleic acid may: a) be blunt ended at both ends; b) have anoverhang at one end and a blunt end at the other; or c) have an overhangat both ends.

One or more nucleotides on the first and/or second strand may bemodified, to form modified nucleotides. One or more of the odd numberednucleotides of the first strand may be modified. One or more of the evennumbered nucleotides of the first strand may be modified by at least asecond modification, wherein the at least second modification isdifferent from the modification on the one or more odd numberednucleotides. At least one of the one or more modified even numberednucleotides may be adjacent to at least one of the one or more modifiedodd numbered nucleotides.

A plurality of odd numbered nucleotides may be modified in the nucleicacid of the invention. A plurality of even numbered nucleotides may bemodified by a second modification. The first strand may compriseadjacent nucleotides that are modified by a common modification. Thefirst strand may also comprise adjacent nucleotides that are modified bya second different modification.

One or more of the odd numbered nucleotides of the second strand may bemodified by a modification that is different to the modification on theodd nucleotides of the first strand and/or one or more of the evennumbered nucleotides of the second strand may be modified by the samemodification on the odd nucleotides of the first strand. At least one ofthe one or more modified even numbered nucleotides of the second strandmay be adjacent to the one or more modified odd numbered nucleotides. Aplurality of odd numbered nucleotides of the second strand may bemodified by a common modification and/or a plurality of even numberednucleotides may be modified by the same modification that is present onthe first strand odd numbered nucleotides. A plurality of odd numberednucleotides may be modified by a second modification, wherein the secondmodification is different to the modification on the first strand oddnumbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified bya common modification, which may be a second modification that isdifferent from the modification on the odd numbered nucleotides of thefirst strand.

In the nucleic acid of the invention, each of the odd numberednucleotides in the first strand and each of the even numberednucleotides in the second strand may be modified with a commonmodification, and each of the even numbered nucleotides may be modifiedin the first strand with a second modification and each of the oddnumbered nucleotides may be modified in the second strand with a secondmodification.

The nucleic acid of the invention may have the modified nucleotides ofthe first strand shifted by at least one nucleotide relative to theunmodified or differently modified nucleotides of the second strand.

The first strand may comprise a sequence selected form the groupconsisting of SEQ ID NO:s 1, 3, 5 and 7 and/or the second strand maycomprise a sequence selected from the group consisting of SEQ ID NO:s 2,4, 6 and 8.

The modification and/or modifications may each and individually beselected from the group consisting of 3′-terminal deoxy-thymine,2′-O-methyl, a 2′-deoxy-modification, a 2′-amino-modification, a2′-alkyl-modification, a morpholino modification, a phosphoramidatemodification, 5′-phosphorothioate group modification, a 5′ phosphate or5′ phosphate mimic modification and a cholesteryl derivative or adodecanoic acid bisdecylamide group modification and/or the modifiednucleotide may be any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide. At least one modificationmay be 2′-O-methyl and/or at least one modification may be 2′-F.

In the nucleic acid of the invention, the inverted nucleotide that isthe terminal nucleotide at the 3′ end of at least one of the firststrand and the second strand that is attached to the adjacent nucleotidevia the 3′ carbon of the terminal nucleotide and the 3′ carbon of theadjacent nucleotide and/or the inverted nucleotide that is the terminalnucleotide at the 5′ end of at least one of the first strand and thesecond strand that is attached to the adjacent nucleotide via the 5′carbon of the terminal nucleotide and the 5′ carbon of the adjacentnucleotide may be a purine.

The nucleic acid of the invention may be conjugated with a ligand.

A nucleic acid of the invention may comprise a phosphorothioate linkagebetween the terminal one, two or three 3′ nucleotides and/or 5′nucleotides of the first and/or the second strand. It may comprise twophosphorothioate linkages between each of the three terminal 3′ andbetween each of the three terminal 5′ nucleotides on the first strand,and two phosphorothioate linkages between the three terminal nucleotidesof the 3′ end of the second strand.

The invention further provides, as a second aspect, a nucleic acid forinhibiting expression of a target gene in a cell, comprising at leastone duplex region that comprises at least a portion of a first strandand at least a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of a RNA transcribed fromsaid target gene to be inhibited and wherein the terminal nucleotide atthe 3′ end of at least one of the first strand and the second strand isan inverted nucleotide and is attached to the adjacent nucleotide viathe 3′ carbon of the terminal nucleotide and the 3′ carbon of theadjacent nucleotide and/or the terminal nucleotide at the 5′ end of atleast one of the first strand and the second strand is an invertednucleotide and is attached to the adjacent nucleotide via the 5′ carbonof the terminal nucleotide and the 5′ carbon of the adjacent nucleotide,and wherein the nucleic acid molecule is conjugated to a ligand.

A ligand for use in the present invention may therefore comprise (i) oneor more N-acetyl galactosamine (GalNac) moieties and derivativesthereof, and (ii) a linker, wherein the linker conjugates the GalNacmoieties to a sequence as defined in any preceding aspects. The linkermay be a bivalent or trivalent or tetravalent branched structure. Thenucleotides may be modified as defined herein.

The ligand may comprise the formula I:

[S—X¹—P—X²]₃-A-X³—  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate).

The present invention therefore additionally provides a conjugatednucleic acid having one of the following structures:

wherein Z represents a nucleic acid as defined herein before.

The ligand may comprise:

The invention also provides a composition comprising a nucleic acid orconjugated nucleic acid as defined herein and a physiologicallyacceptable excipient. The composition may include the followingexcipients:

-   -   i) a cationic lipid, or a pharmaceutically acceptable salt        thereof;    -   ii) a steroid;    -   iii) a phosphatidylethanolamine phospholipid;    -   iv) a PEGylated lipid.

The content of the cationic lipid component in the composition may befrom about 55 mol % to about 65 mol % of the overall lipid content ofthe lipid formulation, preferably about 59 mol % of the overall lipidcontent of the lipid composition.

The composition may comprise;

a cationic lipid having the structure;

a steroid having the structure;

a phosphatidylethanolamine phospholipid having the structure;

and a PEGylated lipid having the structure;

Also provided is a nucleic acid or conjugated nucleic acid according toany aspect of the invention for use in the treatment or prevention of adisease or disorder and/or in the manufacture of a medicament fortreating or preventing a disease or disorder.

The invention provides a method of treating or preventing a disease ordisorder comprising administration of a composition comprising a nucleicacid or conjugated nucleic acid according to any aspect of the inventionto an individual in need of treatment. The nucleic acid may beadministered to the subject subcutaneously or intravenously.

A method of making the nucleic acid according to the invention is alsoincluded.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to a nucleic acid which is double strandedand directed to an expressed RNA transcript of a target gene andcompositions thereof. These nucleic acids can be used in the treatmentof a variety of diseases and disorders where reduced expression oftarget gene products is desirable.

A first aspect of the invention relates to a nucleic acid for inhibitingexpression of a target gene in a cell, comprising at least one duplexregion that comprises at least a portion of a first strand and at leasta portion of a second strand that is at least partially complementary tothe first strand, wherein said first strand is at least partiallycomplementary to at least a portion of a RNA transcribed from saidtarget gene to be inhibited and wherein the terminal nucleotide at the3′ end of at least one of the first strand and the second strand is aninverted nucleotide and is attached to the adjacent nucleotide via the3′ carbon of the terminal nucleotide and the 3′ carbon of the adjacentnucleotide and/or the terminal nucleotide at the 5′ end of at least oneof the first strand and the second strand is an inverted nucleotide andis attached to the adjacent nucleotide via the 5′ carbon of the terminalnucleotide and the 5′ carbon of the adjacent nucleotide.

By nucleic acid it is meant a nucleic acid comprising two strandscomprising nucleotides, that is able to interfere with gene expression.Inhibition may be complete or partial and results in down regulation ofgene expression in a targeted manner. The nucleic acid comprises twoseparate polynucleotide strands; the first strand, which may also be aguide strand; and a second strand, which may also be a passenger strand.The first strand and the second strand may be part of the samepolynucleotide molecule that is self complementary which ‘folds’ to forma double stranded molecule. The nucleic acid may be an siRNA molecule.

The nucleic acid may comprise ribonucleotides, modified ribonucleotides,deoxynucleotides, deoxyribonucleotides, or nucleotide analogues. Thenucleic acid may further comprise a double-stranded nucleic acid portionor duplex region formed by all or a portion of the first strand (alsoknown in the art as a guide strand) and all or a portion of the secondstrand (also known in the art as a passenger strand). The duplex regionis defined as beginning with the first base pair formed between thefirst strand and the second strand and ending with the last base pairformed between the first strand and the second strand, inclusive.

By duplex region it is meant the region in two complementary orsubstantially complementary oligonucleotides that form base pairs withone another, either by Watson-Crick base pairing or any other mannerthat allows for the formation of a duplex between oligonucleotidestrands that are complementary or substantially complementary. Forexample, an oligonucleotide strand having 21 nucleotide units can basepair with another oligonucleotide of 21 nucleotide units, yet only 19nucleotides on each strand are complementary or substantiallycomplementary, such that the “duplex region” consists of 19 base pairs.The remaining base pairs may exist as 5′ and 3′ overhangs, or as singlestranded regions. Further, within the duplex region, 100%complementarity is not required; substantial complementarity isallowable within a duplex region. Substantial complementarity refers tocomplementarity between the strands such that they are capable ofannealing under biological conditions. Techniques to empiricallydetermine if two strands are capable of annealing under biologicalconditions are well known in the art. Alternatively, two strands can besynthesised and added together under biological conditions to determineif they anneal to one another.

The portion of the first strand and second strand that form at least oneduplex region may be fully complementary and are at least partiallycomplementary to each other.

Depending on the length of an nucleic acid, a perfect match in terms ofbase complementarity between the first strand and second strand is notnecessarily required. However, the first and second strands must be ableto hybridise under physiological conditions.

The complementarity between the first strand and second strand in the atleast one duplex region may be perfect in that there are no nucleotidemismatches or additional/deleted nucleotides in either strand.Alternatively, the complementarity may not be perfect. Thecomplementarity may be at least 70%, 75%, 80%, 85%, 90% or 95%.

The first strand and the second strand may each comprise a region ofcomplementarity which comprises at least 15 contiguous nucleotides.

The nucleic acid involves the formation of a duplex region between allor a portion of the first strand and a portion of the target nucleicacid. The portion of the target nucleic acid that forms a duplex regionwith the first strand, defined as beginning with the first base pairformed between the first strand and the target sequence and ending withthe last base pair formed between the first strand and the targetsequence, inclusive, is the target nucleic acid sequence or simply,target sequence. The duplex region formed between the first strand andthe second strand need not be the same as the duplex region formedbetween the first strand and the target sequence. That is, the secondstrand may have a sequence different from the target sequence however,the first strand must be able to form a duplex structure with both thesecond strand and the target sequence.

The complementarity between the first strand and the target sequence maybe perfect (no nucleotide mismatches or additional/deleted nucleotidesin either nucleic acid).

The complementarity between the first strand and the target sequence maynot be perfect. The complementarity may be from about 70% to about 100%.More specifically, the complementarity may be at least 70%, 80%, 85%,90% or 95%, or an intermediate value.

The identity between the first strand and the complementary sequence ofthe target sequence may be from about 75% to about 100%. Morespecifically, the complementarity may be at least 75%, 80%, 85%, 90% or95%, or an intermediate value, provided a nucleic acid is capable ofreducing or inhibiting the expression of a target gene.

A nucleic acid with less than 100% complementarity between the firststrand and the target sequence may be able to reduce the expression of atarget gene to the same level as a nucleic acid with perfectcomplementarity between the first strand and the target sequence.

Alternatively, it may be able to reduce expression of a target gene to alevel that is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of thelevel of expression achieved by the nucleic acid with perfectcomplementarity.

In a further aspect the nucleic acid as described herein may reduce theexpression of a target gene in a cell by at least 10% compared to thelevel observed in the absence of an inhibitor, which may be the nucleicacid. All preferred features of any of the previous aspects also applyto this aspect. In particular, the expression of a target gene in a cellmay be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, andintermediate values, than that observed in the absence of an inhibitor(which may be the nucleic acid).

The nucleic acid may comprise a first strand and a second strand thatare each from 19-25 nucleotides in length. The first strand and thesecond strand may be of different lengths.

The nucleic acid may be 15-25 nucleotide pairs in length. The nucleicacid may be 17-23 nucleotide pairs in length. The nucleic acid may be17-25 nucleotide pairs in length. The nucleic acid may be 23-24nucleotide pairs in length. The nucleic acid may be 19-21 nucleotidepairs in length. The nucleic acid may be 21-23 nucleotide pairs inlength.

The nucleic acid may comprise a duplex region that consists of 19-25nucleotide base pairs. The duplex region may consist of 17, 18, 19, 20,21, 22, 23, 24 or 25 base pairs which may be contiguous.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be attached to the adjacent nucleotide via a phosphate group and beattached via a phosphodiester linkage. The 3′ and/or 5′ invertednucleotide of the first and/or second strand may be attached to theadjacent nucleotide via a phosphorothioate group. The 3′ and/or 5′inverted nucleotide of the first and/or second strand may be attached tothe adjacent nucleotide via a phosphorodithioate group. A sulphur of thephosphorothioate or phosphorodithioate group may be in place of one orboth of the non-linking 0 of a phosphate group. An S may be in place ofa linking 0 of the phosphate group.

The 3′ and/or 5′ inverted nucleotide of the first and/or second strandmay be any nucleotide (i.e. A, G, C or U. Preferably, it may be an A ora G.

The nucleic acid may be blunt ended at both ends; have an overhang atone end and a blunt end at the other end; or have an overhang at bothends.

An “overhang” as used herein has its normal and customary meaning in theart, i.e. a single stranded portion of a nucleic acid that extendsbeyond the terminal nucleotide of a complementary strand in a doublestrand nucleic acid. The term “blunt end” includes double strandednucleic acid whereby both strands terminate at the same position,regardless of whether the terminal nucleotide(s) are base paired. Theterminal nucleotide of a first strand and a second strand at a blunt endmay be base paired. The terminal nucleotide of a first strand and asecond strand at a blunt end may not be paired. The terminal twonucleotides of an first strand and a second strand at a blunt end may bebase paired. The terminal two nucleotides of an first strand and asecond strand at a blunt end may not be paired.

The nucleic acid may have an overhang at one end and a blunt end at theother. The nucleic acid may have an overhang at both ends. The nucleicacid may be blunt ended at both ends. The nucleic acid may be bluntended at the end with the 5′-end of the first strand and the 3′-end ofthe second strand or at the 3′-end of the first strand and the 5′-end ofthe second strand.

The nucleic acid may comprise an overhang at a 3′- or 5′-end. Thenucleic acid may have a 3′-overhang on the first strand. The nucleicacid may have a 3′-overhang on the second strand. The nucleic acid mayhave a 5′-overhang on the first strand. The nucleic acid may have a5′-overhang on the second strand. The nucleic acid may have an overhangat both the 5′-end and 3′-end of the first strand. The nucleic acid mayhave an overhang at both the 5′-end and 3′-end of the second strand. Thenucleic acid may have a 5′ overhang on the first strand and a 3′overhang on the second strand. The nucleic acid may have a 3′ overhangon the first strand and a 5′ overhang on the second strand. The nucleicacid may have a 3′ overhang on the first strand and a 3′ overhang on thesecond strand. The nucleic acid may have a 5′ overhang on the firststrand and a 5′ overhang on the second strand.

An inverted nucleotide may form an overhang at either or both ends ofthe first strand. Ann inverted nucleotide may form an overhang at eitheror both ends of the second strand. An inverted nucleotide may form anoverhang at one and of the first strand and the other end of the secondstrand.

An overhang at the 3′-end or 5′ end of the second strand or the firststrand may be selected from consisting of 1, 2, 3, 4 and 5 nucleotidesin length. Optionally, an overhang may consist of 1 or 2 nucleotides,which may or may not be modified.

The inverted nucleotide may be added as an additional nucleotide to theend of a nucleic acid, i.e. it may form an overhang. Alternatively, theinverted nucleotide may be added in place of a terminal nucleotide ofthe nucleic acid, i.e. it may form a blunt end.

Unmodified polynucleotides, particularly ribonucleotides, may be proneto degradation by cellular nucleases, and, as such,modifications/modified nucleotides may be included in the nucleic acidof the invention.

One or more nucleotides on the second and/or first strand of the nucleicacid of the invention may be modified.

Modifications of the nucleic acid of the present invention generallyprovide a powerful tool in overcoming potential limitations including,but not limited to, in vitro and in vivo stability and bioavailabilityinherent to native RNA molecules. The nucleic acid according to theinvention may be modified by chemical modifications. Modified nucleicacid can also minimise the possibility of inducing interferon activityin humans. Modification can further enhance the functional delivery of anucleic acid to a target cell. The modified nucleic acid of the presentinvention may comprise one or more chemically modified ribonucleotidesof either or both of the first strand or the second strand. Aribonucleotide may comprise a chemical modification of the base, sugaror phosphate moieties. The ribonucleic acid may be modified bysubstitution or insertion with analogues of nucleic acids or bases.

One or more nucleotides of a nucleic acid of the present invention maybe modified. The nucleic acid may comprise at least one modifiednucleotide. The modified nucleotide may be on the first strand. Themodified nucleotide may be in the second strand. The modified nucleotidemay be in the duplex region. The modified nucleotide may be outside theduplex region, i.e., in a single stranded region. The modifiednucleotide may be on the first strand and may be outside the duplexregion. The modified nucleotide may be on the second strand and may beoutside the duplex region. The 3′-terminal nucleotide of the firststrand may be a modified nucleotide. The 3′-terminal nucleotide of thesecond strand may be a modified nucleotide. The 5′-terminal nucleotideof the first strand may be a modified nucleotide. The 5′-terminalnucleotide of the second strand may be a modified nucleotide.

An nucleic acid of the invention may have 1 modified nucleotide or anucleic acid of the invention may have about 2-4 modified nucleotides,or a nucleic acid may have about 4-6 modified nucleotides, about 6-8modified nucleotides, about 8-10 modified nucleotides, about 10-12modified nucleotides, about 12-14 modified nucleotides, about 14-16modified nucleotides about 16-18 modified nucleotides, about 18-20modified nucleotides, about 20-22 modified nucleotides, about 22-24modified nucleotides, 24-26 modified nucleotides or about 26-28 modifiednucleotides. In each case the nucleic acid comprising said modifiednucleotides retains at least 50% of its activity as compared to the samenucleic acid but without said modified nucleotides. The nucleic acid mayretain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or anintermediate value of its activity as compared to the same nucleic acidbut without said modified nucleotides, or may have more than 100% of theactivity of the same nucleotide without said modified nucleotides.

The modified nucleotide may be a purine or a pyrimidine. At least halfof the purines may be modified. At least half of the pyrimidines may bemodified. All of the purines may be modified. All of the pyrimidines maybe modified. The modified nucleotides may be selected from the groupconsisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methylmodified nucleotide, a 2′ modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a nucleotide comprising a 5′-phosphorothioate group, anucleotide comprising a 5′ phosphate or 5′ phosphate mimic and aterminal nucleotide linked to a cholesteryl derivative or a dodecanoicacid bisdecylamide group.

The nucleic acid may comprise a nucleotide comprising a modifiednucleotide, wherein the base is selected from 2-aminoadenosine,2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g.,5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Nucleic acids discussed herein include unmodified RNA as well as RNAwhich has been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, for example as occur naturally in the human body. Modifiednucleotide as used herein refers to a nucleotide in which one or more ofthe components of the nucleic acid, namely sugars, bases, and phosphatemoieties, are different from that which occur in nature. While they arereferred to as modified nucleotides they will of course, because of themodification, include molecules which are not nucleotides, for example apolynucleotide molecules in which the ribophosphate backbone is replacedwith a non-ribophosphate construct that allows hybridisation betweenstrands i.e. the modified nucleotides mimic the ribophosphate backbone.

Many of the modifications described below that occur within a nucleicacid will be repeated within a polynucleotide molecule, such as amodification of a base, or a phosphate moiety, or the a non-linking O ofa phosphate moiety. In some cases the modification will occur at all ofthe possible positions/nucleotides in the polynucleotide but in manycases it will not. A modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal regions, such as at a position ona terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a nucleic acid of the invention or may only occur in asingle strand region of an nucleic acid of the invention. Aphosphorothioate modification at a non-linking O position may only occurat one or both termini, may only occur in a terminal region, e.g., at aposition on a terminal nucleotide or in the last 2, 3, 4 or 5nucleotides of a strand, or may occur in duplex and/or in single strandregions, particularly at termini. The 5′ end or 3′ ends may bephosphorylated.

Stability of an nucleic acid of the invention may be increased byincluding particular bases in overhangs, or by including modifiednucleotides, in single strand overhangs, e.g., in a 5′ or 3′ overhang,or in both. Purine nucleotides may be included in overhangs. All or someof the bases in a 3′ or 5′ overhang may be modified. Modifications caninclude the use of modifications at the 2′ OH group of the ribose sugar,the use of deoxyribonucleotides, instead of ribonucleotides, andmodifications in the phosphate group, such as phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

The 5′- or 3′-overhangs at the first strand, second strand or bothstrands of the dsRNA agent of the invention may be phosphorylated. Insome embodiments, the overhang region contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the first strand, second strand or both strands. In oneembodiment, this 3 ‘-overhang is present in the first strand. In oneembodiment, this 3’-overhang is present in the second strand.

Nucleases can hydrolyze nucleic acid phosphodiester bonds. However,chemical modifications to nucleic acids can confer improved properties,and, can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygens(referred to as linking even if at the 5′ and 3′ terminus of the nucleicacid of the invention);(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar;(iii) replacement of the phosphate moiety with “dephospho” linkers;(iv) modification or replacement of a naturally occurring base;(v) replacement or modification of the ribose-phosphate backbone;(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g., a fluorescently labeled moiety, to either the 3′ or5′ end of RNA.

The terms replacement, modification, alteration, indicates a differencefrom a naturally occurring molecule.

Specific modifications are discussed in more detail below.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulphur. One, each or both non-linking oxygens in thephosphate group can be independently any one of S, Se, B, C, H, N, or OR(R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linkingoxygen with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

A modified nucleotide can include modification of the sugar groups. The2′ hydroxyl group (OH) can be modified or replaced with a number ofdifferent “oxy” or “deoxy” substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleicacids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH2;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino)and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino).

“Deoxy” modifications include hydrogen halo; amino (e.g., NH2;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid);NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl,cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Other substitutents ofcertain embodiments include 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl,2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleotides may contain a sugar suchas arabinose.

Modified nucleotides can also include “abasic” sugars, which lack anucleobase at C-I′. These abasic sugars can further containmodifications at one or more of the constituent sugar atoms.

The 2′ modifications may be used in combination with one or morephosphate linker modifications (e.g., phosphorothioate).

The phosphate group can be replaced by non-phosphorus containingconnectors.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.In certain embodiments, replacements may include themethylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar may be replaced by nucleaseresistant nucleotides.

Examples include the morpholino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNAsurrogates may be used.

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end or the 5′ end or both ends of themolecule. They can include modification or replacement of an entireterminal phosphate or of one or more of the atoms of the phosphategroup. For example, the 3′ and 5′ ends of an oligonucleotide can beconjugated to other functional molecular entities such as labelingmoieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 orCy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron orester). The functional molecular entities can be attached to the sugarthrough a phosphate group and/or a linker. The terminal atom of thelinker can connect to or replace the linking atom of the phosphate groupor the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, thelinker can connect to or replace the terminal atom of a nucleotidesurrogate (e.g., PNAs). These spacers or linkers can include e.g.,—(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—,O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy,amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide,or morpholino, or biotin and fluorescein reagents. The 3′ end can be an—OH group.

Other examples of terminal modifications include dyes, intercalatingagents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid,03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g., biotin), transport/absorption facilitators (e.g.,aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu³⁺ complexes of tetraazamacrocycles).

Alternative or additional terminal modifications can be added for anumber of reasons, including to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogues. Nucleic acids of the invention, on the first or secondstrand, may be 5′ phosphorylated or include a phosphoryl analogue at the5′ prime terminus. 5′-phosphate modifications include those which arecompatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g., RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′vinylphosphonate,5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—),ethoxymethyl, etc., e.g., RP(OH)(O)—O-5′-).

The nucleic acid of the present invention may include one or morephosphorothioate modifications on one or more of the terminal ends ofthe first and/or the second strand. Optionally, each or either end ofthe first strand may comprise one or two or three phosphorothioatemodified nucleotides. Optionally, each or either end of the secondstrand may comprise one or two or three phosphorothioate modifiednucleotides. Optionally, both ends of the first strand and the 5′ end ofthe second strand may comprise two phosphorothioate modifiednucleotides. By phosphorothioate modified nucleotide it is meant thatthe linkage between the nucleotide and the adjacent nucleotide comprisesa phosphorothioate group instead of a standard phosphate group.

Terminal modifications can also be useful for monitoring distribution,and in such cases the groups to be added may include fluorophores, e.g.,fluorescein or an Alexa dye. Terminal modifications can also be usefulfor enhancing uptake, useful modifications for this include cholesterol.Terminal modifications can also be useful for cross-linking an RNA agentto another moiety.

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogues of any of the abovebases and “universal bases” can be employed. Examples include2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N<4>-acetyl cytosine,2-thiocytosine, N6-methyladenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

As used herein, the terms “non-pairing nucleotide analogue” means anucleotide analogue which includes a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analogue is aribonucleotide. In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes withoutlimitation a halogen, alcohol, amine, carboxylic, ester, amide,aldehyde, ketone, ether groups.

Certain moieties may be linked to the 5′ terminus of the first strand orthe second strand and includes abasic ribose moiety, abasic deoxyribosemoiety, modifications abasic ribose and abasic deoxyribose moietiesincluding 2′ O alkyl modifications; inverted abasic ribose and abasicdeoxyribose moieties and modifications thereof, C6-imino-Pi; a mirrornucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotideanalogues including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

The nucleic acids of the invention may be included as one or moreinverted nucleotides, for example inverted thymidine or inverted adenine(for example see Takei, et al., 2002. JBC 277 (26):23800-06).

As used herein, the term “inhibit”, “down-regulate”, or “reduce” withrespect to gene expression means the expression of the gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits (e.g., mRNA), or activity of one or more proteins orprotein subunits or peptides, is reduced below that observed in theabsence of a nucleic acid of the invention or in reference to an siRNAmolecule with no known homology to human transcripts (herein termednon-silencing control). Such control may be conjugated and modified inan analogous manner to the molecule of the invention and delivered intothe target cell by the same route; for example the expression may bereduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less than thatobserved in the absence of an inhibitor (which may be the nucleic acid)or in the presence of a non-silencing control (which may be a nucleicacid that is non-complementary to the target sequence).

The nucleic acid of the present invention may comprise an abasicnucleotide. The term “abasic” as used herein, refers to moieties lackinga base or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative.

The nucleic acid may comprise one or more nucleotides on the secondand/or first strands that are modified. Alternating nucleotides may bemodified, to form modified nucleotides.

Alternating as described herein means to occur one after another in aregular way. In other words, alternating means to occur in turnrepeatedly. For example if one nucleotide is modified, the nextcontiguous nucleotide is not modified and the following contiguousnucleotide is modified and so on. One nucleotide may be modified with afirst modification, the next contiguous nucleotide may be modified witha second modification and the following contiguous nucleotide ismodified with the first modification and so on, where the first andsecond modifications are different.

One or more of the odd numbered nucleotides of the first strand of thenucleic acid of the invention may be modified wherein the first strandis numbered 5′ to 3′. The term “odd numbered” as described herein meansa number not divisible by two. Examples of odd numbers are 1, 3, 5, 7,9, 11 and so on. One or more of the even numbered nucleotides of thefirst strand of the nucleic acid of the invention may be modified,wherein the first strand is numbered 5′ to 3′. The term “even numbered”as described herein means a number which is evenly divisible by two.Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on. One ormore of the odd numbered nucleotides of the second strand of the nucleicacid of the invention may be modified wherein the second strand isnumbered 3′ to 5′. One or more of the even numbered nucleotides of thesecond strand of the nucleic acid of the invention may be modified,wherein the second strand is numbered 3′ to 5′.

One or more nucleotides on the first and/or second strand may bemodified, to form modified nucleotides. One or more of the odd numberednucleotides of the first strand may be modified. One or more of the evennumbered nucleotides of the first strand may be modified by at least asecond modification, wherein the at least second modification isdifferent from the modification on the one or more add nucleotides. Atleast one of the one or more modified even numbered nucleotides may beadjacent to at least one of the one or more modified odd numberednucleotides.

A plurality of odd numbered nucleotides in the first strand may bemodified in the nucleic acid of the invention. A plurality of evennumbered nucleotides in the first strand may be modified by a secondmodification. The first strand may comprise adjacent nucleotides thatare modified by a common modification. The first strand may alsocomprise adjacent nucleotides that are modified by a second differentmodification.

One or more of the odd numbered nucleotides of the second strand may bemodified by a modification that is different to the modification of theodd numbered nucleotides on the first strand and/or one or more of theeven numbered nucleotides of the second strand may be by the samemodification of the odd numbered nucleotides of the first strand. Atleast one of the one or more modified even numbered nucleotides of thesecond strand may be adjacent to the one or more modified odd numberednucleotides. A plurality of odd numbered nucleotides of the secondstrand may be modified by a common modification and/or a plurality ofeven numbered nucleotides may be modified by the same modification thatis present on the first stand odd numbered nucleotides. A plurality ofodd numbered nucleotides on the second strand may be modified by asecond modification, wherein the second modification is different fromthe modification of the first strand odd numbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified bya common modification, which may be a second modification that isdifferent from the modification of the odd numbered nucleotides of thefirst strand.

In the nucleic acid of the invention, each of the odd numberednucleotides in the first strand and each of the even numberednucleotides in the second strand may be modified with a commonmodification and, each of the even numbered nucleotides may be modifiedin the first strand with a second modification and each of the oddnumbered nucleotides may be modified in the second strand with thesecond modification.

The nucleic acid of the invention may have the modified nucleotides ofthe first strand shifted by at least one nucleotide relative to theunmodified or differently modified nucleotides of the second strand.

One or more or each of the odd numbered nucleotides may be modified inthe first strand and one or more or each of the even numberednucleotides may be modified in the second strand. One or more or each ofthe alternating nucleotides on either or both strands may be modified bya second modification. One or more or each of the even numberednucleotides may be modified in the first strand and one or more or eachof the even numbered nucleotides may be modified in the second strand.One or more or each of the alternating nucleotides on either or bothstrands may be modified by a second modification. One or more or each ofthe odd numbered nucleotides may be modified in the first strand and oneor more of the odd numbered nucleotides may be modified in the secondstrand by a common modification. One or more or each of the alternatingnucleotides on either or both strands may be modified by a secondmodification. One or more or each of the even numbered nucleotides maybe modified in the first strand and one or more or each of the oddnumbered nucleotides may be modified in the second strand by a commonmodification. One or more or each of the alternating nucleotides oneither or both strands may be modified by a second modification.

The nucleic acid of the invention may comprise single or double strandedconstructs that comprise at least two regions of alternatingmodifications in one or both of the strands. These alternating regionscan comprise up to about 12 nucleotides but preferably comprise fromabout 3 to about 10 nucleotides. The regions of alternating nucleotidesmay be located at the termini of one or both strands of the nucleic acidof the invention. The nucleic acid may comprise from 4 to about 10nucleotides of alternating nucleotides at each termini (3′ and 5′) andthese regions may be separated by from about 5 to about 12 contiguousunmodified or differently or commonly modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and theeven numbered nucleotides may be modified with a second modification.The second strand may comprise adjacent nucleotides that are modifiedwith a common modification, which may be the same as the modification ofthe odd numbered nucleotides of the first strand. One or morenucleotides of second strand may also be modified with the secondmodification. One or more nucleotides with the second modification maybe adjacent to each other and to nucleotides having a modification thatis the same as the modification of the odd numbered nucleotides of thefirst strand. The first strand may also comprise phosphorothioatelinkages between the two nucleotides at the 3′ end and at the 5′ end.The second strand may comprise a phosphorothioate linkage between thetwo nucleotides at 5′ end. The second strand may also be conjugated to aligand at the 5′ end.

The nucleic acid of the invention may comprise a first strand comprisingadjacent nucleotides that are modified with a common modification. Oneor more of such nucleotides may be adjacent to one or more nucleotideswhich may be modified with a second modification. One or morenucleotides with the second modification may be adjacent. The secondstrand may comprise adjacent nucleotides that are modified with a commonmodification, which may be the same as one of the modifications of oneor more nucleotides of the first strand. One or more nucleotides ofsecond strand may also be modified with the second modification. One ormore nucleotides with the second modification may be adjacent. The firststrand may also comprise phosphorothioate linkages between the twonucleotides at the 5′ end and at the 3′ end. The second strand maycomprise a phosphorothioate linkage between the two nucleotides at 3′end. The second strand may also be conjugated to a ligand at the 5′ end.

The nucleotides numbered (from 5′ to 3′ on the first strand and 3′ and5′ on the second strand) 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and25 may be modified by a modification on the first strand. Thenucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 maybe modified by a second modification on the first strand. Thenucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may bemodified by a modification on the second strand. The nucleotidesnumbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modifiedby a second modification on the second strand. Nucleotides are numberedfor the sake of the nucleic acid of the present invention from 5′ to 3′on the first strand and 3′ and 5′ on the second strand.

The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24may be modified by a modification on the first strand. The nucleotidesnumbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by asecond modification on the first strand. The nucleotides numbered 1, 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification onthe second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22 and 24 may be modified by a second modification on the secondstrand.

Clearly, if the first and/or the second strand are shorter than 25nucleotides in length, such as 19 nucleotides in length, there are nonucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. Theskilled person understands the description above to apply to shorterstrands, accordingly.

One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a common modification.One or more modified nucleotides on the first strand may be paired withmodified nucleotides on the second strand having a differentmodification. One or more modified nucleotides on the first strand maybe paired with unmodified nucleotides on the second strand. One or moremodified nucleotides on the second strand may be paired with unmodifiednucleotides on the first strand. In other words, the alternatingnucleotides can be aligned on the two strands such as, for example, allthe modifications in the alternating regions of the second strand arepaired with identical modifications in the first strand or alternativelythe modifications can be offset by one nucleotide with the commonmodifications in the alternating regions of one strand pairing withdissimilar modifications (i.e. a second or further modification) in theother strand. Another option is to have dissimilar modifications in eachof the strands.

The modifications on the first strand may be shifted by one nucleotiderelative to the modified nucleotides on the second strand, such thatcommon modified nucleotides are not paired with each other.

The modification and/or modifications may each and individually beselected from the group consisting of 3′-terminal deoxy-thymine,2′-O-methyl, a 2′-deoxy-modification, a 2′-amino-modification, a2′-alkyl-modification, a morpholino modification, a phosphoramidatemodification, 5′-phosphorothioate group modification, a 5′ phosphate or5′ phosphate mimic modification and a cholesteryl derivative or adodecanoic acid bisdecylamide group modification and/or the modifiednucleotide may be any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

At least one modification may be 2′-O-methyl and/or at least onemodification may be 2′-F. Further modifications as described herein maybe present on the first and/or second strand.

Throughout the description of the invention, “same or commonmodification” means the same modification to any nucleotide, be that A,G, C or U modified with a group such as such as a methyl group or afluoro group. Is it not taken to mean the same addition on the samenucleotide. For example, 2′F-dU, 2′F-dA, 2′F-dC, 2′F-dG are allconsidered to be the same or common modification, as are 2′-OMe-rU,2′-OMe-rA; 2′-OMe-rC; 2′-OMe-rG. A 2′F modification is a differentmodification to a 2′OMe modification.

Some representative modified nucleic acid sequences of the presentinvention are shown in the examples. These examples are meant to berepresentative and not limiting.

Preferably, the nucleic acid may comprise a modification and the secondor further modification which are each and individually selected fromthe group comprising 2′-O-methyl modification and 2′-F modification. Thenucleic acid may comprise a modification that is 2′-O-methyl (2′OMe)that may be a first modification, and a second modification that is2′-F. The nucleic acid of the invention may also include aphosphorothioate modification and/or a deoxy modification which may bepresent in or between the terminal 1, 2 or 3 nucleotides of each or anyend of each or both strands.

The nucleic acid of the invention may be conjugated to a ligand, to forma conjugate.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. The endosomolytic component may contain a chemical groupwhich undergoes a change in charge or protonation in response to achange in pH. The endosomolytic component may be linear or branched.

Ligands can include therapeutic modifiers, e.g., for enhancing uptake;diagnostic compounds or reporter groups e.g., for monitoringdistribution; cross-linking agents; and nuclease-resistance conferringmoieties. General examples include lipids, steroids, vitamins, sugars,proteins, peptides, polyamines, and peptide mimics. Ligands can includea naturally occurring substance, such as a protein, carbohydrate, orlipid. The ligand may be a recombinant or synthetic molecule.

Ligands can also include targeting groups, e.g. a cell or tissuetargeting agent. The targeting ligand may be a lectin, glycoprotein,lipid or protein.

Other examples of ligands include dyes, intercalating agents,cross-linkers, porphyrins, polycyclic aromatic hydrocarbons, artificialendonucleases or a chelator, lipophilic molecules, alkylating agents,phosphate, amino, mercapto, PEG, MPEG, alkyl, substituted alkyl,radiolabelled markers, enzymes, haptens, transport/absorptionfacilitators, synthetic ribonucelases, or imidazole clusters.

Ligands can be proteins, e.g. glycoproteins or peptides. Ligands mayalso be hormones or hormone receptors. They may also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,or cofactors.

The ligand may be a substance such as a drug which can increase theuptake of the nucleic acid into a cell, for example, by disrupting thecell's cytoskeleton.

The ligand may increase uptake of the nucleic acid into the cell byactivating an inflammatory response. Such ligands include tumournecrosis factor alpha (TNF-alpha), interleukin-1 beta, or gammainterferon.

The ligand may be a lipid or lipid-based molecule. The lipid orlipid-based molecule preferably binds a serum protein. Preferably, thelipid-based ligand binds human serum albumin (HSA). A lipid orlipid-based molecule can increase resistance to degradation of theconjugate, increase targeting or transport into target cell, and/or canadjust binding to a serum protein. A lipid-based ligand can be used tomodulate binding of the conjugate to a target tissue.

The ligand may be a steroid. Preferably, the ligand is cholesterol or acholesterol derivative.

The ligand may be a moiety e.g. a vitamin, which is taken up by a targetcell. Exemplary vitamins include vitamin A, E, K, and the B vitamins.Vitamins may be taken up by a proliferating cell, which may be usefulfor delivering the nucleic acid to cells such as malignant ornon-malignant tumour cells.

The ligand may be a cell-permeation agent, such as a helicalcell-permeation agent. Preferably such an agent is amphipathic.

The ligand may be a peptide or peptidomimetic. A peptidomimetic is amolecule capable of folding into a defined three-dimensional structuresimilar to a natural peptide. The peptide or peptidomimetic ligand mayinclude naturally occurring or modified peptides, or both. A peptide orpeptidomimetic can be a cell permeation peptide, cationic peptide,amphipathic peptide, or hydrophobic peptide. The peptide moiety can be adendrimer peptide, constrained peptide, or crosslinked peptide. Thepeptide moiety can include a hydrophobic membrane translocationsequence. The peptide moiety can be a peptide capable of carrying largepolar molecules such as peptides, oligonucleotides, and proteins acrosscell membranes, e.g. sequences from the HIV Tat protein (GRKKRRQRRRPPQ)and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK). Preferablythe peptide or peptidomimetic is a cell targeting peptide, e.g.arginine-glycine-aspartic acid (RGD)-peptide.

The ligand may be a cell permeation peptide that is capable ofpermeating, for example, a microbial cell or a mammalian cell.

The ligand may be a pharmacokinetic modulator. The pharmacokineticmodulator may be lipophiles, bile acids, steroids, phospholipidanalogues, peptides, protein binding agents, PEG, vitamins, etc.

When two or more ligands are present, the ligands can all have the sameproperties, all have different properties, or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the nucleic acid at the 3′-end, 5′-end, and/orat an internal position. Preferably the ligand is coupled to the nucleicacid via an intervening tether or linker.

In some embodiments the nucleic acid is a double-stranded nucleic acid.In a double-stranded nucleic acid the ligand may be attached to one orboth strands. In some embodiments, a double-stranded nucleic acidcontains a ligand conjugated to the sense strand. In other embodiments,a double-stranded nucleic acid contains a ligand conjugated to theantisense strand.

Ligands can be conjugated to nucleobases, sugar moieties, orinternucleosidic linkages of nucleic acid molecules. Conjugation topurine nucleobases or derivatives thereof can occur at any positionincluding endocyclic and exocyclic atoms. Conjugation to pyrimidinenucleotides or derivatives thereof can also occur at any position.Conjugation to sugar moieties of nucleosides can occur at any carbonatom. Conjugation to internucleosidic linkages may occur at thephosphorus atom of a phosphorus-containing linkage or at an oxygen,nitrogen, or sulphur atom bonded to the phosphorus atom. For amine- oramide-containing internucleosidic linkages, conjugation may occur at thenitrogen atom of the amine or amide or to an adjacent carbon atom.

The ligand is typically a carbohydrate, e.g. a monosaccharide,disaccharide, trisaccharide, tetrasaccharide or polysaccharide. Theligand may be conjugated to the nucleic acid by a linker. The linker maybe a monovalent, bivalent, or trivalent branched linker.

Means for efficient delivery of oligonucleotides, in particular doublestranded nucleic acids of the invention, to cells in vivo is importantand requires specific targeting and substantial protection from theextracellular environment, particularly serum proteins. One method ofachieving specific targeting is to conjugate a targeting moiety orligand to the nucleic acid.

The targeting moiety helps in targeting the nucleic acid to the requiredtarget site and there is a need to conjugate appropriate targetingmoieties for the desired receptor sites for the conjugated molecules tobe taken up by the cells such as by endocytosis. The targeting moiety orligand can be any moiety or ligand that is capable of targeting aspecific receptor.

For example, the Asialoglycoprotein receptor (ASGP-R) is a high capacityreceptor, which is highly abundant on hepatocytes. One of the firstdisclosures of triantennary cluster glycosides was in U.S. Pat. No.5,885,968. Conjugates having three GalNAc ligands and comprisingphosphate groups are known and are described in Dubber et al. (2003).The ASGP-R shows a 50-fold higher affinity forN-Acetyl-D-Galactosylamine (GalNAc) than D-Gal.

Hepatocytes expressing the lectin (asialoglycoprotein receptor; ASGPR),which recognizes specifically terminal β-galactosyl subunits ofglycosylated proteins or other oligosaccharides (P. H. Weigel et. al.,2002) can be used for targeting a drug to the liver by covalent couplingof galactose or galactoseamine to the drug substance (S. Ishibashi, et.al. 1994). Furthermore the binding affinity can be significantlyincreased by the multi-valency effect, which is achieved by therepetition of the targeting unit (E. A. L. Biessen et. al., 1995).

The ASGPR is a mediator for an active endosomal transport of terminalβ-galactosyl containing glycoproteins, thus ASGPR is highly suitable fortargeted delivery of drug candidates like nucleic acid, which have to bedelivered into a cell (Akinc et al.).

The saccharide, which can also be referred to as the ligand, may beselected to have an affinity for at least one type of receptor on atarget cell. In particular, the receptor is on the surface of amammalian liver cell, for example, the hepatic asialoglycoproteinreceptor (ASGP-R).

The saccharide may be selected from N-acetyl galactoseamine, mannose,galactose, glucose, glucosamone and fucose. The saccharide may beN-acetyl galactoseamine (GalNAc).

A ligand for use in the present invention may therefore comprise (i) oneor more N-acetyl galactosamine (GalNac) moieties and derivativesthereof, and (ii) a linker, wherein the linker conjugates the GalNacmoieties to a sequence as defined in any preceding aspects. The linkermay be a bivalent or trivalent or tetravalent branched structure. Thenucleotides may be modified as defined herein.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactoseamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. Both the β-form:2-(Acetylarni no)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably.Preferably, the compounds of the invention comprise the β-form,2-(Acetylarnino)-2-deoxy-β-D-galactopyranose.

The ligand may comprise GalNAc.

The ligand may comprise a compound of formula I:

[S—X¹—P—X²]₃-A-X³—  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH₂)_(n)—O—CH₂— where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate).

In formula I, branching unit “A” branches into three in order toaccommodate the three saccharide ligands. The branching unit iscovalently attached to the ligands and the nucleic acid. The branchingunit may comprise a branched aliphatic group comprising groups selectedfrom alkyl, amide, disulphide, polyethylene glycol, ether, thioether andhydroxyamino groups. The branching unit may comprise groups selectedfrom alkyl and ether groups.

The branching unit A may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; andeach n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; andeach n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein A₁ is O, S, O═O or NH; andeach n independently represents an integer from 1 to 20.

The branching unit may have the structure:

The branching unit may have the structure:

The branching unit may have the structure:

Optionally, the branching unit consists of only a carbon atom.

X³ may be selected from —C₁-C₂₀ alkylene-, —C₂-C₂₀ alkenylene-, analkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀ alkylene)-,—C(O)—C₁-C₂₀ alkylene-, —C₀-C₄ alkylene(Cy)C₀-C₄ alkylene- wherein Cyrepresents a substituted or unsubstituted 5 or 6 membered cycloalkylene,arylene, heterocyclylene or heteroarylene ring, —C₁-C₄alkylene-NHC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)NH—C₁-C₄ alkylene-,—C₁-C₄ alkylene-SC(O)—C₁-C₄ alkylene-, —C₁-C₄ alkylene-C(O)S—C₁-C₄alkylene-, —C₁-C₄ alkylene-OC(O)—C₁-C₄ alkylene-, —C₁-C₄alkylene-C(O)O—C₁-C₄ alkylene-, and —C₁-C₆ alkylene-S—S—C₁-C₆ alkylene-.

X³ may be an alkylene ether of formula —(C₁-C₂₀ alkylene)-O—(C₁-C₂₀alkylene)-. X³ may be an alkylene ether of formula —(C₁-C₂₀alkylene)-O—(C₄-C₂₀ alkylene)-, wherein said (C₄-C₂₀ alkylene) is linkedto Z. X³ may be selected from the group consisting of —CH₂—O—C₃H₆—,—CH₂—O—C₄H₈—, —CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, especially —CH₂—O—C₄H₈—,—CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group islinked to A.

The ligand may comprise a compound of formula (II):

[S—X¹—P—X²]_(n3)-A-X³—  (II)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or an ethylene glycol stem        (—CH₂—CH₂—O)_(m)(—CH₂)₂—    -   wherein m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is C₁-C₈ alkylene;    -   A is a branching unit selected from:

-   -   X³ is a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate)

Branching unit A may have the structure:

Branching unit A may have the structure:

wherein X³ is attached to the nitrogen atom.X³ may be C₁-C₂₀ alkylene. Preferably, X³ is selected from the groupconsisting of —C₃H₆—, —C₄H₈—, —C₆H₁₂— and —C₈H₁₆—, especially —C₄H₈—,—C₆H₁₂— and —C₈H₁₆—.

The ligand may comprise a compound of formula (III):

[S—X¹—P—X²]₃-A-X³  (III)

wherein:

-   -   S represents a saccharide;    -   X¹ represents C₃-C₆ alkylene or an ethylene glycol stem        (—CH₂—CH₂—O)_(m)(—CH₂)₂—    -   wherein m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is an alkylene ether of formula —C₃H₆—O—CH₂—;    -   A is a branching unit;    -   X³ is an alkylene ether of formula selected from the group        consisting of —CH₂—O—CH₂—, —CH₂—O—C₂H₄—, —CH₂—O—C₃H₆—,        —CH₂—O—C₄H₈—, —CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and        —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group is linked to        A,    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate)

The branching unit may comprise carbon. Preferably, the branching unitis carbon.

X³ may be selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and —CH₂—O—C₈H₁₆—.Preferably, X³ is selected from the group consisting of —CH₂—O—C₄H₈—,—CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆.

For any of the above aspects, P represents a modified phosphate group. Pcan be represented by:

wherein Y¹ and Y² each independently represent ═O, ═S, —O⁻, —OH, —SH,—BH₃, —OCH₂CO₂, —OCH₂CO₂R^(x), —OCH₂C(S)OR^(x), and —OR^(x), whereinR^(x) represents C₁-C₆ alkyl and wherein

indicates attachment to the remainder of the compound.

By modified phosphate It is meant a phosphate group wherein one or moreof oxygens is replaced. Examples of modified phosphate groups includephosphorothioate, phosphoroselenates, borano phosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl oraryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulphur. One, each or both non-linkingoxygens in the phosphate group can be independently any one of S, Se, B,C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of a linkingoxygen with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at a terminal oxygen. Replacement of thenon-linking oxygens with nitrogen is possible.

For example, Y¹ may represent —OH and Y² may represent ═O or ═S; or

Y¹ may represent —O⁻ and Y² may represent ═O or ═S;Y¹ may represent ═O and Y² may represent —CH₃, —SH, —OR^(x), or —BH₃Y¹ may represent ═S and Y² may represent —CH₃, OR^(x) or —SH.

It will be understood by the skilled person that in certain instancesthere will be delocalisation between Y¹ and Y².

Preferably, the modified phosphate group is a thiophosphate group.Thiophosphate groups include bithiophosphate (i.e. where Y¹ represents═S and Y² represents —S⁻) and monothiophosphate (i.e. where Y¹represents —O⁻ and Y² represents ═S, or where Y¹ represents ═O and Y²represents —S⁻). Preferably, P is a monothiophosphate. The inventorshave found that conjugates having thiophosphate groups in replacement ofphosphate groups have improved potency and duration of action in vivo.

P may also be an ethylphosphate (i.e. where Y¹ represents ═O and Y²represents OCH₂CH₃).

The saccharide, which can also be referred to as the ligand, may beselected to have an affinity for at least one type of receptor on atarget cell. In particular, the receptor is on the surface of amammalian liver cell, for example, the hepatic asialoglycoproteinreceptor (ASGP-R).

For any of the above aspects, the saccharide may be selected fromN-acetyl with one or more of galactosamine, mannose, galactose, glucose,glucosamine and fructose.

Preferably, the saccharide is two molecules of N-acetyl galactosamine(GalNAc). The compounds of the invention may have 3 ligands which areeach preferably N-acetyl galactosamine.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonlyreferred to in the literature as N-acetyl galactosamine. Reference to“GalNAc” or “N-acetyl galactosamine” includes both the β-form:2-(Acetylamino)-2-deoxy-β-D-galactopyranose and the α-form:2-(Acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments,both the β-form: 2-(Acetylarnino)-2-deoxy-β-D-galactopyranose andα-form: 2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be usedinterchangeably. Preferably, the compounds of the invention comprise theβ-form, 2-(Acetylarnino)-2-deoxy-β-D-galactopyranose.

2-(Acetylamino)-2-deoxy-D-galactopyranose

2-(Acetylamino)-2-deoxy-β-D-galactopyranose

2-(Acetylamino)-2-deoxy-α-D-galactopyranose

For any of the above compounds of formula (III), X¹ may be an ethyleneglycol stem (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein m is 1, 2, or 3. X¹ may be(—CH₂—CH₂—O)(—CH₂)₂—. X¹ may be (—CH₂—CH₂—O)₂(—CH₂)₂—. X¹ may be(—CH₂—CH₂—O)₃(—CH₂)₂—. Preferably, X¹ is (—CH₂—CH₂—O)₂(—CH₂)₂—.Alternatively, X¹ represents C₃-C₆ alkylene. X¹ may be propylene. X¹ maybe butylene. X¹ may be pentylene. X¹ may be hexylene. Preferably thealkyl is a linear alkylene. In particular, X¹ may be butylene.

For compounds of formula (III), X² represents an alkylene ether offormula —C₃H₆—O—CH₂—i.e. C₃ alkoxy methylene, or —CH₂CH₂CH₂OCH₂—.

The present invention therefore additionally provides a conjugatednucleic acid having one of the following structures:

wherein Z represents a nucleic acid as defined herein before.

The invention provides as a further aspect, a nucleic acid forinhibiting expression of a target gene in a cell, comprising at leastone duplex region that comprises at least a portion of a first strandand at least a portion of a second strand that is at least partiallycomplementary to the first strand, wherein said first strand is at leastpartially complementary to at least a portion of a RNA transcribed fromsaid target gene to be inhibited and wherein the terminal nucleotide atthe 3′ end of at least one of the first strand and the second strand isan inverted nucleotide and is attached to the adjacent nucleotide viathe 3′ carbon of the terminal nucleotide and the 3′ carbon of theadjacent nucleotide and/or the terminal nucleotide at the 5′ end of atleast one of the first strand and the second strand is an invertednucleotide and is attached to the adjacent nucleotide via the 5′ carbonof the terminal nucleotide and the 5′ carbon of the adjacent nucleotide,and wherein the nucleic acid molecule is conjugated to a ligand.

The nucleic acid may be conjugated to a ligand as herein described. Thenucleotides of the first and/or second strand may be modified, as hereindescribed.

The ligand may comprise GalNac and may be of the structure set out in 8.

A cleavable linking group is a linker which is stable outside the cellbut is cleaved upon entry into a target cell. Cleavage releases the twoparts the linker is holding together.

In a preferred embodiment, the nucleic acid of the invention comprises acleavable linking group that is cleaved at least 10 times or more,preferably at least 100-fold faster in a target cell or under a firstreference condition (which can, for example, be selected to mimic orrepresent intracellular conditions) than in the blood of a subject, orunder a second reference condition (which can, for example, be selectedto mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g. pH,redox potential, or the presence of degradative molecules. Degradativemolecules include oxidative or reductive enzymes, reductive agents (suchas mercaptans), esterases, endosomes or agents than can create an acidicenvironment, enzymes that can hydrolyze or degrade an acid cleavablelinking group by acting as a general acid, peptidases, and phosphatases.

A cleavable linking group may be a disulphide bond, which is susceptibleto pH.

A linker may include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the target cell. For example, a linker thatincludes an ester group is preferred when a liver cell is the target.Linkers that contain peptide bonds can be used when targeting cells richin peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. In preferred embodiments, useful candidate compoundsare cleaved at least 2, 4, 10 or 100 times faster in the cell (or underin vitro conditions selected to mimic intracellular conditions) ascompared to blood or serum (or under in vitro conditions selected tomimic extracellular conditions).

In one aspect, the cleavable linking group may be a redox cleavablelinking group. The redox cleavable linking group may be a disulphidelinking group.

In one aspect, the linking group may be a phosphate-based cleavablelinking group. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—,—O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—,—S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferredembodiment is —O—P(O)(OH)—O—.

In one aspect, the cleavable linking group may be an acid cleavablelinking group. Preferably the acid cleavable linking group are cleavedin environments where the pH is 6.5 or lower, or are cleaved by agentssuch as enzymes that can act as a general acid. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—; C(O)O, or —OC(O). A preferred embodiment is alinking group where the carbon attached to the oxygen of the ester (thealkoxy group) is an aryl group, substituted alkyl group, or tertiaryalkyl group such as dimethyl pentyl or t-butyl.

In one embodiment, the cleavable linking group may be an ester-basedcleavable linking group. Examples of ester-based cleavable linkinggroups include but are not limited to esters of alkylene, alkenylene andalkynylene groups.

In one embodiment, the cleavable linking group may be a peptide-basedcleavable linking group. Peptide-based cleavable linking groups arepeptide bonds formed between amino acids to yield oligopeptides (e.g.,dipeptides, tripeptides etc.) and polypeptides. The peptide basedcleavage group is generally limited to the peptide bond (i.e., the amidebond) formed between amino acids yielding peptides and proteins and doesnot include the entire amide functional group. Peptide-based cleavablelinking groups have the general formula —NHCHR^(A)C(O)NHCHR^(B)C(O)—,where RA and RB are the R groups of the two adjacent amino acids.

The nucleic acid as described herein may be formulated with a lipid inthe form of a liposome. Such a formulation may be described in the artas a lipoplex. The composition with a lipid/liposome may be used toassist with delivery of the nucleic acid of the invention to the targetcells. The lipid delivery system herein described may be used as analternative to a conjugated ligand. The modifications herein describedmay be present when using the nucleic acid of the invention with a lipiddelivery system or with a ligand conjugate delivery system.

Such a lipoplex may comprise a lipid composition comprising:

-   -   i) a cationic lipid, or a pharmaceutically acceptable salt        thereof;    -   ii) a steroid;    -   iii) a phosphatidylethanolamine phospholipid;    -   iv) a PEGylated lipid.

The cationic lipid may be an amino cationic lipid.

The cationic lipid may have the formula (I):

or a pharmaceutically acceptable salt thereof, wherein:X represents 0, S or NH;R¹ and R² each independently represents a C₄-C₂₂ linear or branchedalkyl chain or a C₄-C₂₂ linear or branched alkenyl chain with one ormore double bonds, wherein the alkyl or alkenyl chain optionallycontains an intervening ester, amide or disulfide;when X represents S or NH, R³ and R⁴ each independently representhydrogen, methyl, ethyl, a mono- or polyamine moiety, or R³ and R⁴together form a heterocyclyl ring;when X represents O, R³ and R⁴ each independently represent hydrogen,methyl, ethyl, a mono- or polyamine moiety, or R³ and R⁴ together form aheterocyclyl ring, or R³ represents hydrogen and R⁴ representsC(NH)(NH2).

The cationic lipid may have the formula (IA):

or a pharmaceutically acceptable salt thereof.

The cationic lipid may have the formula (IB):

or a pharmaceutically acceptable salt thereof.

The content of the cationic lipid component may be from about 55 mol %to about 65 mol % of the overall lipid content of the formulation. Inparticular, the cationic lipid component is about 59 mol % of theoverall lipid content of the formulation.

The formulations further comprise a steroid. the steroid may becholesterol. The content of the steroid may be from about 26 mol % toabout 35 mol % of the overall lipid content of the lipid formulation.More particularly, the content of steroid may be about 30 mol % of theoverall lipid content of the lipid formulation.

The phosphatidylethanolamine phospholipid may be selected from groupconsisting of 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE),1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE),1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE),1,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE) and1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). Thecontent of the phospholipid may be about 10 mol % of the overall lipidcontent of the composition.

The PEGylated lipid may be selected from the group consisting of1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) andC16-Ceramide-PEG. The content of the PEGylated lipid may be about 1 to 5mol % of the overall lipid content of the formulation.

The content of the cationic lipid component in the composition may befrom about 55 mol % to about 65 mol % of the overall lipid content ofthe lipid formulation, preferably about 59 mol % of the overall lipidcontent of the lipid formulation.

The composition may have a molar ratio of the components ofi):ii):iii):iv) selected from 55:34:10:1; 56:33:10:1; 57:32:10:1;58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1;64:25:10:1; and 65:24:10:1.

The composition may comprise a cationic lipid having the structure

a steroid having the structure

a phosphatidylethanolamine phospholipid having the structure

and a PEGylated lipid having the structure

Neutral liposome compositions may be formed from, for example,dimyristoyl phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions may be formedfrom dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomesmay be formed primarily from dioleoyl phosphatidylethanolamine (DOPE).Another type of liposomal composition may be formed fromphosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.Another type is formed from mixtures of phospholipid and/orphosphatidylcholine and/or cholesterol.

A positively charged synthetic cationic lipid,N-[I-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells. DOTMA analogues can also be used to formliposomes.

Derivatives and analogues of lipids described herein may also be used toform liposomes.

A liposome containing a nucleic acid can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The nucleicacid preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the nucleicacid and condense around the nucleic acid to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of nucleic acid.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also be adjusted to favourcondensation.

Nucleic acid formulations may include a surfactant. In one embodiment,the nucleic acid is formulated as an emulsion that includes asurfactant.

A surfactant that is not ionized is a non-ionic surfactant. Examplesinclude non-ionic esters, such as ethylene glycol esters, propyleneglycol esters, glyceryl esters etc., nonionic alkanolamides, and etherssuch as fatty alcohol ethoxylates, propoxylated alcohols, andethoxylated/propoxylated block polymers.

A surfactant that carries a negative charge when dissolved or dispersedin water is an anionic surfactant. Examples include carboxylates, suchas soaps, acyl lactylates, acyl amides of amino acids, esters ofsulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates,sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.

A surfactant that carries a positive charge when dissolved or dispersedin water is a cationic surfactant. Examples include quaternary ammoniumsalts and ethoxylated amines.

A surfactant that has the ability to carry either a positive or negativecharge is an amphoteric surfactant. Examples include acrylic acidderivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

“Micelles” are defined herein as a particular type of molecular assemblyin which amphipathic molecules are arranged in a spherical structuresuch that all the hydrophobic portions of the molecules are directedinward, leaving the hydrophilic portions in contact with the surroundingaqueous phase. The converse arrangement exists if the environment ishydrophobic. A micelle may be formed by mixing an aqueous solution ofthe nucleic acid, an alkali metal alkyl sulphate, and at least onemicelle forming compound.

Exemplary micelle forming compounds include lecithin, hyaluronic acid,pharmaceutically acceptable salts of hyaluronic acid, glycolic acid,lactic acid, chamomile extract, cucumber extract, oleic acid, linoleicacid, linolenic acid, monoolein, monooleates, monolaurates, borage oil,evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine andpharmaceutically acceptable salts thereof, glycerin, polyglycerin,lysine, polylysine, triolein, polyoxyethylene ethers and analoguesthereof, polidocanol alkyl ethers and analogues thereof,chenodeoxycholate, deoxycholate, and mixtures thereof.

Phenol and/or m-cresol may be added to the mixed micellar composition toact as a stabiliser and preservative. An isotonic agent such asglycerine may as be added.

A nucleic acid preparation may be incorporated into a particle such as amicroparticle. Microparticles can be produced by spray-drying,lyophilisation, evaporation, fluid bed drying, vacuum drying, or acombination of these methods.

The present invention also provides pharmaceutical compositionscomprising the nucleic acid or conjugated nucleic acid of the invention.The pharmaceutical compositions may be used as medicaments or asdiagnostic agents, alone or in combination with other agents. Forexample, a nucleic acid or conjugated nucleic acid of the invention canbe combined with a delivery vehicle (e.g., liposomes) and excipients,such as carriers, diluents. Other agents such as preservatives andstabilizers can also be added. Methods for the delivery of a nucleicacid or conjugated nucleic acid are known in the art and within theknowledge of the person skilled in the art.

The nucleic acid or conjugated nucleic acid of the present invention canalso be administered in combination with other therapeutic compounds,either administrated separately or simultaneously, e.g., as a combinedunit dose. The invention also includes a pharmaceutical compositioncomprising a nucleic acid or conjugated nucleic acid according to thepresent invention in a physiologically/pharmaceutically acceptableexcipient, such as a stabilizer, preservative, diluent, buffer, and thelike.

The pharmaceutical composition may be specially formulated foradministration in solid or liquid form. The composition may beformulated for oral administration, parenteral administration(including, for example, subcutaneous, intramuscular, intravenous, orepidural injection), topical application, intravaginal or intrarectaladministration, sublingual administration, ocular administration,transdermal administration, or nasal administration. Delivery usingsubcutaneous or intravenous methods are preferred.

Dosage levels for the medicament and pharmaceutical compositions of theinvention can be determined by those skilled in the art by routineexperimentation. In one embodiment, a unit dose may contain betweenabout 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid.Alternatively, the dose can be from 10 mg/kg to 25 mg/kg body weight, or1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight,or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg bodyweight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kgbody weight. Dosage levels may also be calculated via other parameterssuch as, e.g., body surface area.

The pharmaceutical composition may be a sterile injectable aqueoussuspension or solution, or in a lyophilized form. In one embodiment, thepharmaceutical composition may comprise lyophilized lipoplexes or anaqueous suspension of lipoplexes. The lipoplexes preferably comprises anucleic acid of the present invention. Such lipoplexes may be used todeliver the nucleic acid of the invention to a target cell either invitro or in vivo.

The pharmaceutical compositions and medicaments of the present inventionmay be administered to a mammalian subject in a pharmaceuticallyeffective dose. The mammal may be selected from humans, dogs, cats,horses, cattle, pig, goat, sheep, mouse, rat, hamster and guinea pig.

A further aspect of the invention relates to a nucleic acid orconjugated nucleic acid of the invention or the pharmaceuticalcomposition comprising the nucleic acid or conjugated nucleic acid ofthe invention for use in the treatment or prevention of a disease ordisorder. The invention includes a pharmaceutical composition comprisingone or more RNAi molecules according to the present invention in aphysiologically/pharmaceutically acceptable excipient, such as astabiliser, preservative, diluent, buffer and the like.

The pharmaceutical composition may be a sterile injectable aqueoussuspension or solution, or in a lyophilised form.

Pharmaceutically acceptable compositions may comprise atherapeutically-effective amount of one or more nucleic acid(s) in anyembodiment according to the invention, taken alone or formulated withone or more pharmaceutically acceptable carriers, excipient and/ordiluents.

Examples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

Stabilisers may be agents that stabilise the nucleic acid agent, forexample a protein that can complex with the nucleic acid, chelators(e.g. EDTA), salts, RNAse inhibitors, and DNAse inhibitors.

In some cases it is desirable to slow the absorption of the drug fromsubcutaneous or intramuscular injection in order to prolong the effectof a drug. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

The nucleic acid described herein may be capable of inhibiting theexpression of a target gene in a cell. The nucleic acid described hereinmay be capable of partially inhibiting the expression of a target genein a cell. Inhibition may be complete, i.e. 0% of the expression levelof target gene expression in the absence of the nucleic acid of theinvention. Inhibition of target gene expression may be partial, i.e. itmay be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% oftarget gene expression in the absence of a nucleic acid of theinvention. Inhibition may last 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or up to 3months, when used in a subject, such as a human subject. The nucleicacid or composition comprising the nucleic acid composition may be foruse once, every week, every two weeks, every three weeks, every fourweeks, every five weeks, every six weeks, every seven weeks, or everyeight weeks. The nucleic acid may be for use subcutaneously,intravenously or using any other application routes such as oral, rectalor intraperitoneal.

In cells and/or subjects treated with or receiving the nucleic acid ofthe present invention, the target gene expression may be inhibitedcompared to untreated cells and/or subjects by at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or100%. The level of inhibition may allow treatment of a diseaseassociated with target gene expression or overexpression, or may allowfurther investigation into the functions of the target gene product.

The target gene may be Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR,RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RASgene, MEKK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA(p21) gene, MYBgene, JU gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C,Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene,survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase IIalpha gene, mutations in the p73 gene, mutations in the p21(WAF I/CIPI)gene, mutations in the p27(KIPI) gene, mutations in the PPM ID gene,mutations in the RAS gene, mutations in the caveolin I gene, mutationsin the MIB I gene, mutations in the MTAI gene, mutations in the M68gene, mutations in tumor suppressor genes, and mutations in the p53tumor suppressor gene.

A further aspect of the invention relates to nucleic acid of theinvention in the manufacture of a medicament for treating or preventinga disease or disorder.

Also included in the invention is a method of treating or preventing adisease or disorder comprising administration of a pharmaceuticalcomposition comprising a nucleic acid or conjugated nucleic acid asdescribed herein, to an individual in need of treatment. The nucleicacid composition may be administered twice every week, once every week,every two weeks, every three weeks, every four weeks, every five weeks,every six weeks, every seven weeks, or every eight weeks. The nucleicacid or conjugated nucleic acid may be administered to the subjectsubcutaneously, intravenously or using any other application routes suchas oral, rectal or intraperitoneal.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a nucleic acid agent. The maintenance dose ordoses can be the same or lower than the initial dose, e.g., one-halfless of the initial dose. The maintenance doses are, for example,administered no more than once every 2, 5, 10, or 30 days. The treatmentregimen may last for a period of time which will vary depending upon thenature of the particular disease, its severity and the overall conditionof the patient.

In one embodiment, the composition includes a plurality of nucleic acidagent species. In another embodiment, the nucleic acid agent species hassequences that are non-overlapping and non-adjacent to another specieswith respect to a naturally occurring target sequence. In anotherembodiment, the plurality of nucleic acid agent species is specific fordifferent naturally occurring target genes. In another embodiment, thenucleic acid agent is allele specific.

The nucleic acid or conjugated nucleic acid of the present invention canalso be administered or for use in combination with other therapeuticcompounds, either administered separately or simultaneously, e.g. as acombined unit dose.

The nucleic acid or conjugated nucleic acid of the present invention canbe produced using routine methods in the art including chemicallysynthesis or expressing the nucleic acid either in vitro (e.g., run offtranscription) or in vivo. For example, using solid phase chemicalsynthesis or using an expression vector. In one embodiment, theexpression vector can produce the nucleic acid of the invention in atarget cell. Methods for the synthesis of the nucleic acid describedherein are known to persons skilled in the art.

Another aspect of the invention includes any nucleic acid, method, useor composition disclosed herein, wherein there is no terminalphosphorothioate in the nucleic acid.

Another aspect of the invention includes any nucleic acid, method, useor composition disclosed herein, wherein the terminal nucleotide islocated at the 3′ end of at least one of the first strand and the secondstrand, or both.

Another aspect of the invention includes any nucleic acid, method, useor composition disclosed herein, wherein the ligand does not contain aphosphorothioate, such as a nucleic acid according conjugated to aGalnac moiety which does not contain phosphorothioates.

The invention will now be described with reference to the followingnon-limiting figures and examples in which:

FIG. 1 shows the 3′-3′ and 5′-5′ linkages used to form an invertednucleotide;

FIG. 2 shows siRNA sequences targeting TMPRSS6 with inverted RNAnucleotides at 3′-ends of first and second strand;

FIGS. 3 and 4 show the serum stability of ivR-modified siRNAs;

FIG. 5 shows the in vitro knockdown activity of ivR-modified siRNAs;

FIG. 6 shows the in vitro knockdown activity of selected ivR-modifiedsiRNAs;

FIG. 7 shows the structure of an example of a GalNac ligand referred toherein;

FIG. 8 shows siRNA sequences targeting TMPRSS6 withphosphorothioate-linked inverted RNA nucleotides;

FIG. 9 shows in vitro activity siRNAs with phosphorothioate-linkedinverted A and G RNA nucleotides at terminal 3′ positions;

FIG. 10 shows the serum stability of different siRNA duplexes containinginverted RNA nucleotides at both 3′-ends;

FIG. 11 shows in vitro activity of siRNAs targeting TMPRSS6 withinverted RNA nucleotides at terminal 3′ positions;

FIG. 12 shows in vitro activity of siRNAs targeting TMPRSS6 withinverted RNA nucleotides at terminal 3′ positions;

FIG. 13 shows the in vitro activity of a GalNAc-conjugated siRNAtargeting TMPRSS6 and containing inverted RNA nucleotides at terminal 3′positions after liposomal transfection;

FIG. 14 shows the serum stability of different siRNA duplexes targetingALDH2 and containing inverted RNA nucleotides at both 3′-ends;

FIG. 15 shows the in vitro activity of siRNAs targeting ALDH2 withinverted A, U, C and G RNA nucleotides at 3′-overhang positions;

FIG. 16 shows the in vitro activity of siRNAs targeting ALDH2 withinverted A, U, C and G RNA nucleotides at terminal 3′ positions;

FIG. 17 shows the serum stability of different GalNAc-siRNA conjugatestargeting ALDH2 and containing inverted RNA nucleotides;

FIG. 18 shows the in vitro activity of GalNAc-conjugated siRNAstargeting ALDH2 with inverted RNA nucleotides at terminal 3′ positionsafter receptor-mediated uptake in mouse primary hepatocytes;

FIG. 19 shows the in vivo activity of GalNAc-conjugated siRNAs targetingALDH2 with ivA at the first strand 3′-end in mice;

FIGS. 20 and 21 show in vitro activity of GalNAc-conjugated siRNAsagainst ALDH2 (STS22006) containing inverted RNA nucleotides in additionto terminal nucleotides.

FIGS. 22 and 23 show in vitro activity of GalNAc-conjugated siRNAsagainst ALDH2 (STS22009) containing inverted RNA nucleotides in additionto terminal nucleotides.

FIGS. 24 and 25 show in vitro activity of GalNAc-conjugated siRNAsagainst TTR containing inverted RNA nucleotides in addition to terminalnucleotides.

FIGS. 26 and 27 show in vitro activity of GalNAc-conjugated siRNAsagainst ALDH2 containing inverted RNA nucleotides at 3′-ends instead ofthe last nucleotide.

FIGS. 28 and 29 show in vivo activity of different modified variants ofthe GalNAc-siRNA conjugates STS22006 and STS22009 in mice.

FIG. 30 shows siRNA sequences with 5′-5′-linked ribonucleotides.

FIG. 31 shows serum stability of siRNAs containing 5′-5′-linkedribonucleotides at the 5′-end of the first strand.

FIG. 32 shows in vitro activity of siRNAs with 5′-5′-linkedribonucleotides at the 5′-end of the first strand.

FIG. 33 shows in vitro activity of siRNAs with 5′-5′-linkedribonucleotides at the 5′-end of the second strand.

EXAMPLES Example 1

siRNA Modification: Using Inverted Nucleotides.

The terminal nucleotide at the 3′ end of an oligonucleotide strand canbe attached to the adjacent nucleotide via the 3′ carbon of the terminalnucleotide and the 3′ carbon of the adjacent nucleotide to form a3′-3′-inverted nucleotide (FIG. 1A). Likewise, the terminal nucleotideat the 5′ end of an oligonucleotide strand can be attached to theadjacent nucleotide via the 5′ carbon of the terminal nucleotide and the5′ carbon of the adjacent nucleotide to form a 5′-5′-inverted nucleotide(FIG. 1B). 5′-3′, 3′-3′ and 5′-5′ phosphodiester linkages are shown inFIG. 1.

Example 2

siRNA Modification: Synthesis of siRNA with Inverted Nucleotides.

All Oligonucleotides were either obtained from a commercialoligonucleotide manufacturer (Eurogentech, Belgium) or synthesized on anAKTA oligopilot synthesizer using standard phosphoramidite chemistry.Commercially available solid support and 2′O-Methyl RNA phosphoramidtes,2′Fluoro DNA phosphoramidites (all standard protection) and commerciallyavailable long trebler phosphoramidite (Glen research) were used.Synthesis was performed using 0.1 M solutions of the phosphoramidite indry acetonitrile and benzylthiotetrazole (BTT) was used as activator(0.3M in acetonitrile). All other reagents were commercially availablestandard reagents.

Conjugation of the GalNac synthon (ST23) or treblers ST41 and ST43 wasachieved by coupling of the respective phosphoramidite to the 5′end ofthe oligochain under standard phosphoramidite coupling conditions.Phosphorothioates were introduced using standard commercially availablethiolation reagents (EDITH, Link technologies).

ST23 is a GalNac C4 phosphoramidite (structure components as below)

Itrb is as follows:

Long trebler (Itrb)

ST41 is as follows (and as described in WO2017/174657):

ST43 is as follows (and as described in WO2017/174657):

The single strands were cleaved off the CPG by using Methylamine. WhereTBDMS protected RNA nucleosides were used, additional treatment withTEA*3HF was performed to remove the silyl protection, as known in theart. The resulting crude oligonucleotide was purified by Ionexchangechromatography (Resource Q, 6 mL, GE Healthcare) on a AKTA Pure HPLCSystem using a Sodium chloride gradient. Product containing fractionswere pooled, desalted on a size exclusion column (Zetadex, EMP Biotech)and lyophilised.

For Duplexation, equimolar amounts of the respective single strands weredissolved in water and heated to 80° C. for 5 min. After cooling theresulting Duplex was lyophilised.

Example 3

siRNAs containing different inverted RNA bases in their 3′-terminalpositions were tested for serum stability.

All siRNAs are modified by alternating 2′-OMe/2′-F in both strands, suchthat every 2′-OMe modified nucleotide on the first strand is paired witha 2′-F modified nucleotide on the second strand. TMP70 comprises twoterminal phosphorothioates at 5′- and 3′-ends of both strands. TMP71-74are modified by terminal phosphorothioates at 5′- and 3′-ends and oneadditional inverted nucleotide (A, U, C, G) at their 3′-ends. Incontrast, TMP75-78 each have two phosphorothioate at the 5′-ends, nophosphorothioate at the 3′-ends and one additional inverted nucleotide(A, U, C, G) at the 3′-ends. Inverted RNA nucleotides are attached via aphosphodiester linkage.

Serum stability of ivR-modified siRNAs was tested. “w/o FBS” and “UT”indicates untreated samples. “FBS” indicates siRNA duplexes which wereincubated at 5 μM final concentration with 50% FBS for 3 d,phenol/chloroform-extracted and precipitated with Ethanol. Samples wereanalyzed on 20% TBE polyacrylamide gels in native gel electrophoresis.TMP75 (which includes an inverted A) and TMP78 (which includes aninverted G) are more stable than TMP70.

Data are shown in FIGS. 2-4.

Example 4

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalyzed using an siRNA against TMPRSS6. TMP7O-TMP74 containphosphorothioates at all termini, whereas TMP75-TMP78 do not containterminal phosphorothioates at the 3′-ends of both strands. Inverted RNAnucleotides are present in addition to the terminal nucleotide asinverted A (TMP71, TMP75), inverted U (TMP72, TMP76), inverted C (TMP73, TMP77) and inverted G (TMP74, TMP78). These siRNAs were tested forknockdown of the target gene in vitro. A non-related siRNA (PTEN) and anon-targeting siRNA (Luci) were included as controls. All testedvariants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 0.1 and 1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Each bar represents mean±SD of three technical replicates.

Knockdown activity of ivR-modified siRNAs in vitro was tested. A Hep3Bcell line was seeded at a density of 150,000 cells per 6-well.Experimental conditions: 0.1 and 1 nM siRNA, 1 μg/ml Atufect, lysis 48hpt. All variants were found to be equally active in vitro.

Data are shown in FIGS. 2 and 5.

Example 5

The influence of inverted A and G RNA nucleotides at terminal 3′positions was analyzed using an siRNA against TMPRSS6. TMP70 containsphosphorothioates at all termini, whereas TMP75 contains ivA and TMP78contains ivG at the 3′-ends of both first and second strand. At theseends, ivA and ivG substitute for terminal phosphorothioates and arepresent in addition to the terminal nucleotide of the respectivestrands. The siRNA were tested for target knockdown in vitro. Anon-related siRNA (PTEN) and a non-targeting siRNA (Luci) were includedas controls. All tested variants show comparable activity under thetested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 5 to 0.00016 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Each bar represents mean±SD of three technical replicates.

Data are shown in FIGS. 2 and 6.

Example 6

The influence of inverted A and G RNA nucleotides at terminal 3′positions was analysed using an siRNA against TMPRSS6. TMP70 containseach two phosphorothioate linkages at all termini, whereas TMP82 andTMP83 contain ivA (TMP82) and ivG (TMP83) at the T-end of the firststrand and at the 3′-end of the second strand. Both inverted nucleotidesare present in addition to the terminal nucleotide of the respectivestrands and are linked via a phosphorothioate bond. A non-related siRNA(PTEN) and a non-targeting siRNA (Luci) were included as controls. Alltested variants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR. Theresults are shown in FIG. 9. Each bar represents mean±SD from threetechnical replicates.

Data are shown in FIGS. 8 and 9.

Example 7

Different siRNA duplexes containing inverted RNA nucleotides at both3′-ends were tested for serum stability. TMP84-TMP87 contain invertedRNA in addition to the last nucleotide in the second strand and insteadof the last nucleotide in the first strand. TMP88-TMP91 contain invertedRNA in addition to the last nucleotide in the first strand and insteadof the last nucleotide in the second strand. All inverted RNAnucleotides substitute for terminally used phosphorothioates. In thedesign of TMP84-TMP87, ivA and ivG confer higher stability to the testedsequence than ivU and ivC (part A). In the design of TMP88-TMP91, thereis no influence of base identity on duplex stability (part B).

“UT” indicates untreated samples. “FBS” indicates siRNA duplexes whichwere incubated at 5 μM final concentration with 50% FBS for 3 d,phenol/chloroform-extracted and precipitated with Ethanol. Samples wereanalysed on 20% TBE polyacrylamide gels in native gel electrophoresisand results are shown in FIG. 10.

Sequences are set out in Table 1.

TABLE 1 Different siRNA duplexes containing inverted RNA nucleotides at both 3′-ends. sequence and chemistry Duplextop: first strand,  ID bottom: second strand, both 5′-3′ TMP70mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU (ps)fG(ps)mAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG (ps)mU(ps)fU TMP84mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fG ivAfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mUfU ivG TMP85mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fG ivUfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mUfU ivG TMP86mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fG ivCfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mUfU ivG TMP87mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fG ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mUfU ivG TMP88mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mU ivA TMP89mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mU ivU TMP90mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mU ivC TMP91mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU fGmA ivGfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG mU ivG mA, mU, mC, mG-2′-OMeRNA fA, fU, fC, fG-2′-F RNA ivA, ivU, ivC, ivG-inverted RNA (3′-3′)(ps)-phosphorothioate

Example 8

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalysed using an siRNA against TMPRSS6. Sequences are set out inTable 1. TMP70 contains phosphorothioates at all termini, whereasTMP84-TMP87 contain ivG at the 3′-end of the second strand. The invertedRNA nucleotide is present in addition to the last nucleotide andsubstitutes for two terminal phosphorothioates. At the first strand3′-end, ivA (TMP84), ivU (TMP85), ivC (TMP86) and ivG (TMP87) weretested. These inverted RNA nucleotides were added instead of theterminal nucleotide and substitute for phosphorothioates. A non-relatedsiRNA (PTEN) and a non-targeting siRNA (Luci) were included as controls.All tested variants show comparable activity under the testedconditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 11. Each bar represents mean±SD of threetechnical replicates.

Example 9

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalysed using an siRNA against TMPRSS6. The sequences are set out inTable 1. TMP70 contains phosphorothioates at all termini, whereasTMP88-TMP91 contain ivG at the 3′-end of the first strand. The invertedRNA nucleotide is present in addition to the last nucleotide andsubstitutes for two phosphorothioates. At the second strand 3′-end, ivA(TMP88), ivU (TMP89), ivC (TMP90) and ivG (TMP91) were tested. Theseinverted RNA nucleotides were added instead of the terminal nucleotideand substitute for phosphorothioates. A non-related siRNA (PTEN) and anon-targeting siRNA (Luci) were included as controls. All testedvariants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 1 nM and 0.1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 12. Each bar represents mean±SD of threetechnical replicates.

Example 10

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalysed using a GalNAc-siRNA conjugate targeting TMPRSS6 in liposomaltransfections. STS12009-L4 contains phosphorothioates at allnon-conjugated termini, whereas the tested variants contain an invertedRNA nucleotide at the 3′-ends of both first and second strand. Theinverted RNA is present in addition to the last nucleotide andsubstitutes for two terminal phosphorothioates (STS12009V10-L4 and-V11-L4) or is used in addition to two terminal phosphorothioates(STS12009V29-L4 and STS12009V30-L4). Inverted A (STS12009V10-L4 and-V29-L4) and inverted G (STS12009V11-L4 and -V30-L4) were used. Alltested variants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 5 nM to 0.0016 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand TMPRSS6 and Actin mRNA levels were determined by Taqman qRT-PCR.Each bar represents mean±SD of three technical replicates.

Sequences are listed in Table 2 and results are shown in FIG. 13.

TABLE 2 GaINAc-siRNA conjugates targeting TMPRSS6 sequence were used to investigate the influence ofinverted RNA nucleotides at terminal 3′ positions. Sequence chemistryDuplex Top: first strand, ID bottom: second strand, both 5′-3′STS12009L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCf AmGfGmU(ps)fG(ps)mA[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGm CfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUSTS12009V10L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCf AmGfGmUfGmA ivA[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGm CfUmUfCmUfUmCfUmGfGmUfU ivASTS12009V11L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCf AmGfGmUfGmA ivG[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGm CfUmUfCmUfUmCfUmGfGmUfU ivGSTS12009V29L4 mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivA [ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivA STS12009V30L4mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCf AmGfGmU(ps)fG(ps)mA ivG[ST23(ps)]3 ST41(ps)fUmCfAmCfCmUfGm CfUmUfCmUfUmCfUmGfG(ps)mU(ps)fUivGmA, mU, mC, mG-2′OMe RNA fA, fU, fC, fG-2′F RNA ivA, ivG-inverted RNA(3′-3′) (ps)-phosphorothioate

Example 11

Different siRNA duplexes targeting ALDH2 and containing inverted RNAnucleotides at both 3′-ends were tested for serum stability. ALD02-ALD05contain inverted RNA in addition to the last nucleotide in first andsecond strand. ALD06-ALD09 contain inverted RNA instead of the lastnucleotide in first and second strand. All inverted RNA nucleotidessubstitute for terminally used phosphorothioates. In both designs, ivAand ivG confer higher stability to the tested sequence than ivU and ivC.

“UT” indicates untreated samples. “FBS” indicates siRNA duplexes whichwere incubated at 5 μM concentration with 50% FBS for 3 d,phenol/chloroform-extracted and precipitated with Ethanol. Samples wereanalysed on 20% TBE polyacrylamide gels in native gel electrophoresisand results are shown in FIG. 14.

Sequences are shown in Table 3.

TABLE 3 Different siRNA duplexes containing inverted RNA nucleotides at both 3′-ends, where each sequence targets ALDH2. sequence and chemistryDuplex top: first strand, ID bottom: second strand, both 5′-3′ ALD01mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC (ps)fG(ps)mGfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA (ps)mU(ps)fU ALD02mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fGmG ivAfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mUfU ivA ALD03mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fGmG ivUfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mUfU ivU ALD04mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fGmG ivCfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mUfU ivC ALD05mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fGmG ivGfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mUfU ivG ALD06mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fG ivAfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mU ivA ALD07mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fG ivUfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mU ivU ALD08mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fG ivCfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mU ivC ALD09mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC fG ivGfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA mU ivG mA, mU, mC, mG-2′-OMeRNA fA, fU, fC, fG-2′-F RNA ivA, ivU, ivC, ivG-inverted RNA (3′-3′)(ps)-phosphorothioate

Example 12

The influence of inverted A, U, C and G RNA nucleotides at 3′-overhangpositions was analysed using an siRNA against ALDH2. Sequences are setout in Table 3. ALD01 contains phosphorothioates at all termini, whereasALD02-ALD05 contain ivA (ALD01), ivU (ALD03), ivC (ALD04) and ivG(ALD05) at the 3′-end of the first strand and at the 3′-end of thesecond strand. Both inverted nucleotides are present in addition to theterminal nucleotide of the respective strands and substitute forterminal phosphorothioates. A non-related siRNA (PTEN) and anon-targeting siRNA (Luci) were included as controls. All testedvariants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 0.1 nM and 1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand ALDH2 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 15. Each bar represents mean±SD of threetechnical replicates.

Example 13

The influence of inverted A, U, C and G RNA nucleotides at terminal 3′positions was analysed using an siRNA against ALDH2. Sequences are setout in Table 3. ALD01 contains phosphorothioates at all termini, whereasALD06-ALD09 contain ivA (ALD06), ivU (ALD07), ivC (ALD08) and ivG(ALD09) at the 3′-end of the first strand and at the 3′-end of thesecond strand. Both inverted nucleotides are present instead of theterminal nucleotide of the respective strands and substitute forterminal phosphorothioates. A non-related siRNA (PTEN) and anon-targeting siRNA (Luci) were included as controls. All testedvariants show comparable activity under the tested conditions.

The experiment was conducted in Hep3B. Cells were seeded at a density of150,000 cells per 6-well, transfected with 0.1 nM and 1 nM siRNA and 1μg/ml Atufect after 24 h and lysed after 48 h. Total RNA was extractedand ALDH2 and Actin mRNA levels were determined by Taqman qRT-PCR.Results are shown in FIG. 16. Each bar represents mean±SD of threetechnical replicates.

Example 14

Different GalNAc-siRNA conjugates containing inverted RNA nucleotideswere tested for serum stability. STS22002L6 contains phosphorothioatesat all non-conjugated ends, whereas STS22002V1 L6 and STS22002V2L6contain inverted RNA nucleotides at the second strand 3′-end, where thenucleotide is present instead of the last nucleotide. STS22002V3L6 and-V4L6 contain inverted RNA nucleotides at the first strand 3′-end, wherethe nucleotide is present in addition to the last nucleotide. ivA wasused in STS22002V1L6 and -V3L6, whereas ivG was used in STS22002V2L6 and-V4L6. All inverted RNA nucleotides substitute for terminally usedphosphorothioates. STS22002V1 L6 and -V2L6 are slightly more stable thanthe other variants tested here.

“UT” indicates untreated samples. “FBS” indicates GalNAc-siRNAconjugates which were incubated at 5 μM concentration with 50% FBS for 3d, phenol/chloroform-extracted and precipitated with Ethanol. Sampleswere analysed on 20% TBE polyacrylamide gels in native gelelectrophoresis and results are shown in FIG. 17.

Sequences are set out in Table 4.

TABLE 4 Different GaINAc-siRNA conjugatesof a ALDH2 targeting sequence,  containing inverted RNA nucleotides.Sequence chemistry Duplex Top: first strand, IDbottom: second strand, both 5′-3′ STS22002L6mA(ps)fA(ps)mUfGmUfUmUfmCfCmUfGmCfUmGf AmC(ps)fG(ps)mG[ST23(ps)]3 ST43(ps)fCmCfGmUfCmAfGmCfA mGfGmAfAmAfAmCfA(ps)mU(ps)fUSTS22002V1L6 mA(ps)fA(ps)mUfGmUfUmUfmCfCmUfGmCfUmGf AmC(ps)fG(ps)mG[ST23(ps)]3 ST43(ps)fCmCfGmUfCmAfGmCfA mGfGmAfAmAfAmCfAmUfU ivASTS22002V2L6 mA(ps)fA(ps)mUfGmUfUmUfmCfCmUfGmCfUmGf AmC(ps)fG(ps)mG[ST23(ps)]3 ST43(ps)fCmCfGmUfCmAfGmCfA mGfGmAfAmAfAmCfAmUfU ivGSTS22002V3L6 mA(ps)fA(ps)mUfGmUfUmUfmCfCmUfGmCfUmGf AmCfGmG ivA[ST23(ps)]3 ST43(ps)fCmCfGmUfCmAfGmCfA mGfGmAfAmAfAmCfA(ps)mU(ps)fUSTS22002V4L6 mA(ps)fA(ps)mUfGmUfUmUfmCfCmUfGmCfUmGf AmCfGmG ivG[ST23(ps)]3 ST43(ps)fCmCfGmUfCmAfGmCfA mGfGmAfAmAfAmCfA(ps)mU(ps)fU mA,mU, mC, mG-2′OMe RNA fA, fU, fC, fG-2′F RNA ivA, ivG-inverted RNA(3′-3′) (ps)-phosphorothioate

Example 14

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalysed using a GalNAc-siRNA conjugate targeting ALDH2 byreceptor-mediated uptake in mouse primary hepatocytes. The sequences areset out in Table 4. STS22002L6 contains phosphorothioates at allnon-conjugated ends, whereas STS22002V1L6 and

STS22002V2L6 contain inverted RNA nucleotides at the second strand3′-end, where the nucleotide is present instead of the last nucleotide.STS22002V3L6 and -V4L6 contain inverted RNA nucleotides at the firststrand 3′-end, where the nucleotide is present in addition to the lastnucleotide. ivA was used in STS22002V1L6 and -V3L6, whereas ivG was usedin STS22002V2L6 and -V4L6. All inverted RNA nucleotides substitute forterminally used phosphorothioates. All tested variants show comparableactivity.

The experiment was conducted in primary mouse hepatocytes. Cells wereseeded at a density of 20,000 cells per 96-well, treated with 125 nM to0.04 nM siRNA conjugate directly after plating and lysed after 24 h.Total RNA was extracted and ALDH2 and Actin mRNA levels were determinedby Taqman qRT-PCR. Results are shown in FIG. 18. Each bar representsmean±SD of three technical replicates.

Example 15

The influence of ivA at the first strand 3′-end was analysed in vivo inmice. Therefore, GalNAc-siRNA conjugates targeting ALDH2 were used.Sequences are set out in Table 4. STS22002L6 contains phosphorothioatesat all non-conjugated termini, whereas STS22002V3L6 contains ivA at thefirst strand 3′-end in addition to the last nucleotide.

C57BL/6 male mice were subcutaneously treated with 10 mg/kg and 3 mg/kgGalNAc conjugate. Liver sections were prepared 7 days after treatment,RNA was extracted from the tissue and ALDH2 and ApoB mRNA levels wereanalysed by Taqman qRT-PCR. Results are shown in FIG. 19. Each barrepresents mean±SD of six animals.

Example 16

In vitro activity of GalNAc-conjugated siRNAs against ALDH2 (STS22006)containing inverted RNA nucleotides in addition to terminal nucleotides.

The influence of inverted RNA nucleotides at terminal positions wasanalyzed using GalNAc-siRNA conjugates targeting ALDH2 afterreceptor-mediated uptake in mouse primary hepatocytes. STS22006L6contains phosphorothioates at all non-conjugated ends, whereasSTS22006V7L6 contains one ivA at the first strand 3′-end andSTS22006V8L6 contains one ivA at the second strand 3′-end. STS22006V9L6contains each one ivA at the first strand and second strand 3′-ends. Thenamed conjugates contain a GalNAc moiety at the second strand 5′-end.STS22006V10L35 contains a GalNAc moiety at the first strand 3′-end witheach one ivA at the second strand 5′- and 3′-ends. All siRNA conjugatesdescribed here contain ivA instead of the terminal nucleotide andinstead of terminal phosphorothioates.

The experiment was conducted in primary mouse hepatocytes. Cells wereseeded at a density of 20,000 cells per 96-well, treated with 100, 10and 1 nM siRNA conjugate directly after plating and lysed after 24 h.Total RNA was extracted and ALDH2 and PTEN mRNA levels were determinedby Taqman qRT-PCR. Each bar represents mean±SD of three technicalreplicates

Data is shown in FIGS. 20 and 21

Example 17

In vitro activity of GalNAc-conjugated siRNAs against ALDH2 (STS22009)containing inverted RNA nucleotides in addition to terminal nucleotides.

The influence of inverted RNA nucleotides at terminal positions wasanalyzed using GalNAc-siRNA conjugates targeting ALDH2 afterreceptor-mediated uptake in mouse primary hepatocytes. STS22009L6contains phosphorothioates at all non-conjugated ends, whereasSTS22009V3L6 contains one ivA at the first strand 3′-end andSTS22009V4L6 contains one ivA at the second strand 3′-end. STS22009V5L6contains each one ivA at the first strand and second strand 3′-ends. Thenamed conjugates contain a GalNAc moiety at the second strand 5′-end.STS22009V6L35 contains a GalNAc moiety at the first strand 3′-end witheach one ivA at the second strand 5′- and 3′-ends. All siRNA conjugatesdescribed here contain ivA instead of the terminal nucleotide andinstead of terminal phosphorothioates.

The experiment was conducted in primary mouse hepatocytes. Cells wereseeded at a density of 20,000 cells per 96-well, treated with 100, 10and 1 nM siRNA conjugate directly after plating and lysed after 24 h.Total RNA was extracted and ALDH2 and PTEN mRNA levels were determinedby Taqman qRT-PCR. Each bar represents mean±SD of three technicalreplicates

Data is shown in FIGS. 22 and 23

Example 18

In vitro activity of GalNAc-conjugated siRNAs against TTR containinginverted RNA nucleotides in addition to terminal nucleotides.

The influence of inverted RNA nucleotides at terminal positions wasanalyzed using GalNAc-siRNA conjugates targeting TTR afterreceptor-mediated uptake in mouse primary hepatocytes. STS16001L1contains phosphorothioates at all non-conjugated ends, whereasSTS16001V11L1 contains one ivA at the first strand 3′-end andSTS16001V12L1 contains one ivA at the second strand 3′-end.STS16001V13L1 contains each one ivA at the first strand and secondstrand 3′-ends. The named conjugates contain a GalNAc moiety at thesecond strand 5′-end. STS16001V14L35 contains a GalNAc moiety at thefirst strand 3′-end with each one ivA at the second strand 5′- and3′-ends. All siRNA conjugates described here contain ivA instead of theterminal nucleotide and instead of terminal phosphorothioates.

The experiment was conducted in primary mouse hepatocytes. Cells wereseeded at a density of 20,000 cells per 96-well, treated with 10, 1 and0.1 nM siRNA conjugate directly after plating and lysed after 24 h.Total RNA was extracted and TTR and PTEN mRNA levels were determined byTaqman qRT-PCR. Each bar represents mean±SD of three technicalreplicates.

Data is shown in FIGS. 24 and 25

Example 19

In vitro activity of GalNAc-conjugated siRNAs against ALDH2 containinginverted RNA nucleotides at 3′-ends instead of the last nucleotide.

The influence of inverted RNA nucleotides at terminal 3′ positions wasanalyzed using GalNAc-siRNA conjugates targeting ALDH2 afterreceptor-mediated uptake in mouse primary hepatocytes. STS22002L6contains phosphorohioates at all non-conjugated ends, whereasSTS22002V8L6 contains one ivA at the first strand 3′-end instead of thelast nucleotide and instead of phosphorothioates. STS22002V9L6 containseach one ivA at the first and second strand 3′-ends instead of therespective last nucleotides and terminal phosphorothioates.STS22002V10L6 contains one ivA at the first strand 3′-end in addition tothe last nucleotide and one ivA at the second strand 3′-end instead ofthe last nucleotide. Both ivA-containing ends are not stabilized byterminal phosphorothioates and the siRNA is conjugated to a GalNAcmoiety which does not contain phosphorothioates.

The experiment was conducted in primary mouse hepatocytes. Cells wereseeded at a density of 20,000 cells per 96-well, treated with 100, 10and 1 nM siRNA conjugate directly after plating and lysed after 24 h.Total RNA was extracted and ALDH2 and PTEN mRNA levels were determinedby Taqman qRT-PCR. Each bar represents mean±SD of three technicalreplicates.

Data is shown in FIGS. 26 and 27

Example 20

Different modified variants of the GalNAc-siRNA conjugates STS22006 andSTS22009 were analyzed for knockdown activity in vivo. “V1” variantscontain a different 2′-0Me/2′-F modification pattern in the secondstrand and an ivA nucleotide at the 3′-end of the second strand,substituting for the last nucleotide and for terminal phosphorothioatesat this end. “V2” additionally contains a different 2′-OMe/2′-Fmodification pattern in the first strand.

C57BL/6 male mice were subcutaneously treated with 3 mg/kg and 1 mg/kgGalNAc conjugate. Liver sections were prepared 9 days after treatment,RNA was extracted from the tissue and ALDH2 and ApoB mRNA levels wereanalyzed by Taqman qRT-PCR. Each bar represents mean±SD of six animals.Statistical analysis is based on Kruskal-Wallis test with Dunn'smultiple comparisons test against control group (PBS).

Data is shown in FIGS. 28 and 29.

Example 21

Serum stability of siRNA-conjugates (X0258-261) with non-cleavableGalNAc linker at the 5′-end of the second strand and 3′ phosphorylatedivR substituting the first nucleotide at the 5′-end of the first strandin comparison to stable X0139 and less stabilized positive (Juk) controlfor nuclease degradation.

The siRNA conjugates were incubated for 4 hours (4h) or 3 days (3d) in50% FBS at 37° C. or left untreated (Oh). After incubation, RNA wasextracted by phenol/chloroform/isoamyl alcohol extraction. Degradationwas visualized by TBE-Polyacrylamid gel electrophoresis and staining ofRNA with SYBRGold.

Data are shown in FIGS. 30 and 31.

Example 22

Target gene expression in primary murine hepatocytes 24 h followingtreatment with TTR-siRNA conjugates with non-cleavable GalNAc-cluster atthe 5′-end of the second strand and with one 3′-phosphorylated invertedribonucleotide at the 5′-position of the first strand, withoutstabilizing phosphorothioate linkages between the three terminalnucleotides at that end (X0258-261), in comparison to a non-targetingGalNAc-siRNA (Luc), and a positive control (X0139) at indicatedconcentrations or cells left untreated (UT).

The experiment was conducted in murine primary hepatocytes. Cells wereseeded at a density of 30,000 cells per 96-well and treated withsiRNA-conjugates at concentrations ranging from 10 nM to 0.0001 nM. 24 hpost treatment cells were lysed and RNA was extracted. Transcript levelsof TTR and housekeeping mRNA (PTEN) were quantified by TaqMan analysis.Each bar represents mean±SD of three technical replicates.

Data are shown in FIGS. 30 and 32.

Example 23

Target gene expression in primary murine hepatocytes 24 h followingtreatment with TTR-siRNA with a GalNAc-cluster at the 3′-end of thefirst strand and one inverted ribonucleotide as an overhang at the5′-position of the second strand replacing the two stabilizingphosphorothioate linkages between the first three nucleotides at thisend (X0264-267), in comparison to a non-targeting GalNAc-siRNA (Luc),and a positive control (X0107) at indicated concentrations or leftuntreated (UT).

The experiment was conducted in murine primary hepatocytes. Cells wereseeded at a density of 30,000 cells per 96-well and treated withsiRNA-conjugates at concentrations ranging from 10 nM to 0.001 nM. 24 hpost treatment cells were lysed and RNA was extracted. Transcriptslevels of TTR and housekeeping mRNA (PTEN) were quantified by TaqMananalysis. Each bar represents mean±SD of three technical replicates.

Data are shown in FIGS. 30 and 33.

Example 24

Additional example compounds were synthesised by the methods describedbelow and methods known to the person skilled in the art. Assembly ofthe oligonucleotide chain and linker building blocks was performed bysolid phase synthesis applying phosphoramidte methodology. GalNAcconjugation was achieved by peptide bond formation of aGalNAc-carboxylic acid building block to the prior assembled andpurified oligonucleotide having the necessary number of amino modifiedlinker building blocks attached.

Oligonucleotide synthesis, deprotection and purification followedstandard procedures that are known in the art.

Oligonucleotides were synthesized on an AKTA oligopilot synthesizerusing standard phosphoramidite chemistry. Commercially available solidsupport and 2′O-Methyl RNA phosphoramidites, 2′Fluoro, 2′Deoxy RNAphosphoramidites, 2′TBDMS RNA phosphoramidites (all standard protection,ChemGenes, LinkTech) and commercially available long treblerphosphoramidite (Glen research) and 3′-Amino Modifier TFA Amino C-6 lcaaCPG 500 Å (CPG supported GlyC3Am(TFA)) was purchased from ChemGenes.Per-acetylated galactose amine 8 is commercially available. Phosphategenerating agent Bis-cyanoethyl-N,N-diisopropyl phosphoramidite waspurchased from ChemGenes.

Ancillary reagents were purchased from EMP Biotech. Synthesis wasperformed using a 0.1 M solution of the phosphoramidite in dryacetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3Min acetonitrile). Coupling time was 15 min. A Cap/OX/Cap or Cap/Thio/Capcycle was applied (Cap: Ac2O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.1MI2 in pyridine/H2O). Phosphorothioates were introduced using standardcommercially available thiolation reagent (EDITH, Link technologies).DMT cleavage was achieved by treatment with 3% dichloroacetic acid intoluene. Upon completion of the programmed synthesis cycles adiethylamine (DEA) wash was performed. All oligonucleotides weresynthesized in DMT-off mode.

GlyC3Am(TFA)-solid support is:

Synthesis of Compounds 2 to 10 and ST13

Compounds 2 to 5 and (S)-DMT-Serinol(TFA)-phosphoramidite 7 weresynthesised according to literature published methods (Hoevelmann et al.Chem. Sci., 2016, 7, 128-135).

i) ethyl trifluoroacetate, NEt3, MeOH, 0° C., 16h, 5: 90%, ii) DMTCI,pyridine, 0° C., 16h, 64% over two steps, iii) LiBH4, EtOH/THF (1/1,v/v), 0° C., 1h, 76%, iv) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, EtNiPr2, CH2Cl2, 56%, v) succinic anhydride, DMAP,pyridine, RT, 16h, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), RT, 18h,29% (26 μmol/g loading).

(S)-4-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-(2,2,2-trifluoroacetamido)propoxy)-4-oxobutanoicacid (6)

To a solution of 5 in pyridine was added succinic anhydride, followed byDMAP. The resulting mixture was stirred at room temperature overnight.All starting material was consumed, as judged by TLC. The reaction wasconcentrated. The crude material was chromatographed in silica gel usinga gradient 0% to 5% methanol in DCM (+1% triethylamine) to afford 1.33 gof 6 (yield=38%). m/z (ESI−): 588.2 (100%), (calcd. forC30H29F3NO8-[M−H]− 588.6). 1H-NMR: (400 MHz, CDCl3) δ [ppm]=7.94 (d, 1H,NH), 7.39-7.36 (m, 2H, CHaryl), 7.29-7.25 (m, 7H, CHaryl), 6.82-6.79 (m,4H, CHaryl), 4.51-4.47 (m, 1H), 4.31-4.24 (m, 2H), 3.77 (s, 6H,2×DMTr-OMe), 3.66-3.60 (m, 16H, HNEt3+), 3.26-3.25 (m, 2H), 2.97-2.81(m, 20H, NEt3), 2.50-2.41 (4H, m), 1.48-1.45 (m, 26H, HNEt3+), 1.24-1.18(m, 29H, NEt3).

(S)-DMT-Serinol(TFA)-succinate-lcaa-CPG (10)

The (S)-DMT-Serinol(TFA)-succinate (159 mg, 270 umol) and HBTU (113 mg,299 umol) were dissolved in CH3CN (10 mL). Diisopropylethylamine (DIPEA,94 μL, 540 umol) was added to the solution, and the mixture was swirledfor 2 min followed by addition native amino-lcaa-CPG (500 A, 3 g, aminecontent: 136 umol/g). The suspension was gently shaken at roomtemperature on a wrist-action shaker for 16h then filtered, and washedwith DCM and EtOH. The solid support was dried under vacuum for 2 h. Theunreacted amines on the support were capped by stirring with aceticanhydride/lutidine/N-methylimidazole at room temperature. The washing ofthe support was repeated as above. The solid was dried under vacuum toyield solid support 10 (3 g, 26 umol/g loading).

Synthesis of GalNAc synthon 9 was performed as described in Nair et al.J. Am. Chem. Soc., 2014, 136 (49), pp 16958-16961.

(vii) TMSOTf, DCM, hexenol, viii) RuCl3, NalO4, DCM, CH3CN, H2O, 46%over two steps.

Synthesis of ST13(Ac)9 was achieved by following methods as described inNair et al. J. Am. Chem. Soc., 2014, 136 (49), pp 16958-16961. Finaldeacetylation to yield ST13 was achieved by treating ST13(Ac)9 withsodium methoxide in methanol.

Trimeric GalNAc Synthon (ST13).

ST13(Ac)9 (3150 mg, 1.570 mmol) was dissolved in Methanol (100 ml) andsodium methoxide (5.4M, 227 mg, 1.512 mmol, 280 μL) was added (viasyringe) at room temperature. The resulting mixture was stirred at for 1h. Acetonitrile was added (75 ml) and the reaction mixture wasconcentrated under reduced pressure. m/z (ESI+): 814.5 (100%), (calcd.for C73H131N10O302+[M+2H]2+814.5). 1H NMR (400 MHz, DMSO-d6)δ[ppm]=7.91-7.72 (m, 9H, NH), 7.08 (s, 1H, NH), 4.90 (d, 3H), 4.77 (m,3H), 4.20 (d; 3H), 3.70-3.64 (m, 9H), 3.57-3.40 (br, 30H, incl. res.H2O), 3.26 (m, 6H), 3.03-3.01 (m, 12H), 2.27-2.25 (m, 6H), 2.07-2.03 (m,10H), 1.89-1.85 (t, 2H), 1.78 (s, 9H), 1.52-1.41 (m, 22H); 1.21 (m,12H).

Synthesis of Oligonucleotides

Oligonucleotide synthesis of 3′trivalent tree-like GalNAc-clusterconjugated oligonucleotides commenced using commercially availableGlyC3Am-solid support as in the example compound 168. Phosphoramiditesynthesis coupling cycle consisting of 1) DMT-removal, 2) chainelongation using the required DMT-masked phosphoramidite, 3) capping ofnon-elongated oligonucleotide chains, followed by oxidation of theP(III) to P(V) either by Iodine or EDITH (if phosphorothioate linkagewas desired) and again capping (Cap/Ox/Cap or Cap/Thio/Cap) was repeateduntil full length of the product was reached. Upon completion of chainelongation, the protective DMT group of the last coupled amiditebuilding block was removed, as in step 1) of the phosphoramiditesynthesis cycle.

Oligonucleotide synthesis of multiple 3′ mono-GalNAc conjugatedoligonucleotides was commenced using(S)-DMT-Serinol(TFA)-succinate-lcaa-CPG (10) as in example compound 87.A second and third (S)-DMT-serinol(TFA) was coupled in the first andsecond cycle to the serinol(TFA)-CPG in order to make the precursorcompound 11 for the example compound 87. Afterwards, phosphoramiditesynthesis cycle was applied using 5′-DMT-2′OMe-RNA or 5′-DMT-2′F-DNAphosphoramidites until full length of the product was reached. Uponcompletion of chain elongation, the protective DMT group of the lastcoupled amidite building block was removed, as in step 1) of thephosphoramidite synthesis cycle.

Finally, the respective oligonucleotides were off the CPG and set freefrom additional protective groups by 40% aq. methylamine treatment. Thistreatment also liberated the amino function in the Serinol(TFA) andGlyC3Am(TFA) building block. The crude products were then purified eachby ion exchange chromatography (Resource Q, 6 mL, GE Healthcare) on anAKTA Pure HPLC System using a sodium chloride gradient. Productcontaining fractions were pooled, desalted on a size exclusion column(Zetadex, EMP Biotech) and lyophilized to yield the precursoroligonucleotides 1 or 11 for further GalNAc conjugation.

All final single stranded products were analysed by AEX-HPLC to provetheir purity. Identity of the respective single stranded products(non-modified, amino-modified precursors or GalNAc conjugatedoligonucleotides) was proved by LC-MS analysis.

Conjugation to Single Stranded Oligonucleotides

Conjugated Singles Strands SEQ ID 168, 180, 182, 184 and 186

Conjugation of the GalNac synthon (ST13) was achieved by coupling to the3′-amino function of the respective oligonucleotide strand (1) using apeptide coupling reagent. Therefore, the respective amino-modifiedprecursor molecule was dissolved in H₂O (500 OD/mL) and DMSO (DMSO/H2O,2/1, v/v) was added, followed by DIPEA (2.5% of total volume). In aseparate reaction vessel pre-activation of the trimeric-GalNAc-synthon(ST13) was performed by reacting 2 eq. of the carboxylic acid componentwith 2 eq. of HBTU in presence of 8 eq. DIPEA in DMSO. After 2 min thepre-activated compound ST13 was added to the solution of the respectiveamino-modified precursor molecule 1. After 30 min the reaction progresswas monitored by LCMS or AEX-HPLC. Upon completion of the conjugationreaction the crude product was precipitated by addition of 10x iPrOH and0.1x 2M NaCl and harvested by centrifugation and decantation. Theresulting pellet was dissolved in H2O and finally purified again byanion exchange and size exclusion chromatography and lyophilised.

Conjugated Singles Strands 87, 107, 171, 173, 175, 177 and 179

Conjugation of the GalNac synthon (9) was achieved by coupling to theserinol-amino function of the respective oligonucleotide strand 11 usinga peptide coupling reagent. Therefore, the respective amino-modifiedprecursor molecule 11 was dissolved in H2O (500 OD/mL) and DMSO(DMSO/H2O, 2/1, v/v) was added, followed by DIPEA (2.5% of totalvolume). In a separate reaction vessel pre-activation of theGalN(Ac4)-C4-acid (9) was performed by reacting 2 eq. (per aminofunction in the amino-modified precursor oligonucleotide 11) of thecarboxylic acid component with 2 eq. of HBTU in presence of 8 eq. DIPEAin DMSO. After 2 min the pre-activated compound 9 was added to thesolution of the respective amino-modified precursor molecule. After 30min the reaction progress was monitored by LCMS or AEX-HPLC. Uponcompletion of the conjugation reaction the crude product wasprecipitated by addition of 10x iPrOH and 0.1x 2M NaCl and harvested bycentrifugation and decantation. To set free the acetylated hydroxylgroups in the GalNAc moieties the resulting pellet was dissolved in 40%MeNH2 (1 mL per 500 OD) and after 15 min at RT diluted in H2O (1:10) andfinally purified again by anion exchange and size exclusionchromatography and lyophilised to yield the final product 12.

Ser(GN) Conjugated singles strands 87, 107, 171, 173, 175, 177 and 179is a GalNAc-C4 building block attached to serinol derived linker moiety:

wherein the O--- is the linkage between the oxygen atom and e.g. H,phosphoroate linkage or phosphorothioate linkage.

Serinol derived linker moieties may be based on serinol in anystereochemistry i.e. derived from L-serine isomer, D-serine isomer, aracemic serine or other combination of isomers. In a preferred aspect ofthe invention, the serinol-GalNAc moiety (SerGN) has the followingstereochemistry:

i.e. is based on an (S)-serinol-amidite or (S)-serinol succinate solidsupported building block derived from L-serine isomer.

Double Strand Formation

Individual single strands were dissolved in a concentration of 60 OD/mLin H2O. Both individual oligonucleotide solutions were added together ina reaction vessel. For easier reaction monitoring a titration wasperformed. The first strand was added in 25% excess over the secondstrand as determined by UV-absorption at 260 nm. The reaction mixturewas heated to 80° C. for 5 min and then slowly cooled to RT. Doublestrand formation was monitored by ion pairing reverse phase HPLC. Fromthe UV-area of the residual single strand the needed amount of thesecond strand was calculated and added to the reaction mixture. Thereaction was heated to 80° C. again and slowly cooled to RT. Thisprocedure was repeated until less than 10% of residual single strand wasdetected.

SEQ ID Name Sequence (5′-3′)   1 TMPJH01AmAfAmCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA   2 TMPJH01BfUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU   3 TMPJH40AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA   4 TMPJH40BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU   5 TMPJH41AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivA   6 TMPJH41BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivA   7 TMPJH42AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivU   8 TMPJH42BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivU   9 TMPJH43AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivC  10 TMPJH43BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivC  11 TMPJH44AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivG  12 TMPJH44BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivG  13 TMPJH45AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivA  14 TMPJH45BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivA  15 TMPJH46AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivU  16 TMPJH46BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivU  17 TMPJH47AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivC  18 TMPJH47BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivC  19 TMPJH48AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  20 TMPJH48BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  21 TMP82AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivA  22 TMP82BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivA  23 TMP83AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA(ps)ivG  24 TMP83BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU(ps)ivG  25 TMP84AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivA  26 TMP84BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  27 TMP85AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivU  28 TMP85BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  29 TMP86AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivC  30 TMP86BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  31 TMP87AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfG ivG  32 TMP87BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  33 TMP88AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  34 TMP88BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivA  35 TMP89AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  36 TMP89BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivU  37 TMP90AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  38 TMP90BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivC  39 TMP91AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  40 TMP91BfU(ps)mC(ps)fAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmU ivG  41 STS12009L4AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA  42 STS12009L4B[ST23(ps)]3 ST41(ps) fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU  43STS12009V10L4A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivA  44STS12009V10L4B[ST23(ps)]3 ST41(ps) fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivA  45STS12009V11L4A mA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmUfGmA ivG  46STS12009V11L4B[ST23(ps)]3 ST41(ps) fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfGmUfU ivG  47STS12009V29L4AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivA  48STS12009V29L4B[ST23(ps)]3 ST41(ps) fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivA 49 STS12009V30L4AmA(ps)fA(ps)mCfCmAfGmAfAmGfAmAfGmCfAmGfGmU(ps)fG(ps)mA ivG  50STS12009V30L4B[ST23(ps)]3 ST41(ps) fUmCfAmCfCmUfGmCfUmUfCmUfUmCfUmGfG(ps)mU(ps)fU ivG 51 ALD01A mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG  52ALD01B fC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU  53 ALD02AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivA  54 ALD02BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivA  55 ALD03AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivU  56 ALD03BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivU  57 ALD04AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivC  58 ALD04BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivC  59 ALD05AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivG  60 ALD05BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmUfU ivG  61 ALD06AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivA  62 ALD06BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA  63 ALD07AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivU  64 ALD07BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivU  65 ALD08AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivC  66 ALD08BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivC  67 ALD09AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivG  68 ALD09BfC(ps)mC(ps)fGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivG  69 STS22002L6AmA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG  70 STS22002L6B[ST23(ps)]3 ST43(ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU  71STS22002V1L6A mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG  72STS22002V1L6B[ST23(ps)]3 ST43(ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA  73STS22002V2L6A mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC(ps)fG(ps)mG  74STS22002V2L6B[ST23(ps)]3 ST43(ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivG  75STS22002V3L6A mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivA  76STS22002V3L6B[ST23(ps)]3 ST43(ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU  77STS22002V4L6A mA(ps)fA(ps)mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivG  78STS22002V4L6B[ST23(ps)]3 ST43(ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA(ps)mU(ps)fU  79STS22006L6AmU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU (ps) fU (ps) mC  80STS22006L6B[ST23(ps)]3 ST43(ps) fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfA (ps) mG (ps) fA 81 STS22006V7L6AmU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU (ps) fU (ps) mC  82STS22006V7L6B[ST23(ps)]3 ST43 (ps) fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfAmGfA ivA  83STS22006V8L6AmU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU (ps) fU (ps) mC ivA  84STS22006V8L6B[ST23(ps)]3 ST43 (ps) fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfA (ps) mG (ps) fA 85 STS22006V9L6A mU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmUfUmC ivA 86 STS22006V9L6B[ST23(ps)]3 ST43(ps) fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfAmGfA ivA  87STS22006V10L35AmU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU (ps) fU (ps) mC [(ps)Ser(GN)]3 88 STS22006V10L35B ivA fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfAmGfA ivA  89STS22009L6AmA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU (ps) fC (ps) mU  90STS22009L6B[ST23(ps)]3 ST43 (ps) fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfC (ps) mA (ps) fU 91 STS22009V3L6AmA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU (ps) fC (ps) mU  92STS22009V3L6B[ST23(ps)]3 ST43 (ps) fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfCmAfU ivA  93STS22009V4L6A mA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmUfCmU ivA  94STS22009V4L6B[ST23(ps)]3 ST43 (ps) fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfC (ps) mA (ps) fU 95 STS22009V5L6A mA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmUfCmU ivA 96 STS22009V5L6B[ST23(ps)]3 ST43 (ps) fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfCmAfU ivA  97STS22009V6L6AmA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU (ps) fC (ps) mU[(ps)Ser(GN)]3 98 STS22009V6L6B ivA fAmGfAmAfGmAfUmCfCmUfCmGfGmCfUmAfCmAfU ivA  99STS16001L1AmU (ps) fU (ps) mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG (ps) fU (ps) mU 100STS16001L1B[ST23(ps)]3 ltrb(ps) fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU (ps) mA (ps) fA101 STS16001V11L1AmU (ps) fU (ps) mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG (ps) fU (ps) mU 102STS16001V11L1B[ST23(ps)]3 ltrb(ps) fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfUmAfA ivA 103STS16001V12L1AmU (ps) fU (ps) mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmGfUmU ivA 104STS16001V12L1B[ST23(ps)]3 ltrb(ps) fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU (ps) mA (ps) fA105 STS16001V13L1AmU (ps) fU (ps) mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmGfUmU ivA 106STS16001V13L1B[ST23(ps)]3 ltrb(ps) fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfUmAfA ivA 107STS16001V14L35AmU (ps) fU (ps) mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG (ps) fU (ps) mU[(ps)Ser(GN)]3108 STS16001V14L35B ivA fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfUmAfA ivA109 STS22002L6AmA (ps) fA (ps) mUfGmUfUmUfUmCfCmUfGmCfUmGfAmC (ps) fG (ps) mG 110STS22002L6B[ST23(ps)]3 ST43 (ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA (ps) mU (ps) fU111 STS22002V8L6A mA (ps) fA (ps) mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivA112 STS22002V8L6B[ST23(ps)]3 ST43 (ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfA (ps) mU (ps) fU113 STS22002V9L6A mA (ps) fA (ps) mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfG ivA114 STS22002V9L6B[ST23(ps)]3 ST43 (ps) fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA 115STS22002V10L6A mA (ps) fA (ps) mUfGmUfUmUfUmCfCmUfGmCfUmGfAmCfGmG ivA116 STS22002V10L6B[ST23(ps)]3 ST43 fCmCfGmUfCmAfGmCfAmGfGmAfAmAfAmCfAmU ivA 117STS22006V1L6AmU (ps) fC (ps) mUfUmCfUmUfAmAfAmCfUmGfAmGfUmU (ps) fU (ps) mC 118STS22006V1L6B[ST23(ps)]3 ST43(ps) fGmAfAmAfCmUfCmAfGmUfUmUfAmAfGmAfAmG ivA 119STS22009V1L6AmA (ps) fU (ps) mGfUmAfGmCfCmGfAmGfGmAfUmCfUmU (ps) fC (ps) mU 120STS22009V1L6B[ST23(ps)]3 ST43(ps) mAmGmAmAmGmAfUfCfCmUmCmGmGmCmUmAmCmA ivA 121STS22009V2L6AmA (ps) fU (ps) mGmUmAmGmCmCmGmAmGmGmAfUmCmUmU (ps) mC (ps) mU 122STS22009V2L6B[ST23(ps)]3 ST43(ps) mAmGmAmAmGmAfUfCfCmUmCmGmGmCmUmAmCmA ivA 123TMPJH01A AACCAGAAGAAGCAGGUGA 124 TMPJH01B UCACCUGCUUCUUCUGGUU 125TMPJH41A AACCAGAAGAAGCAGGUGAA 126 TMPJH41B UCACCUGCUUCUUCUGGUUA 127TMPJH42A AACCAGAAGAAGCAGGUGAU 128 TMPJH42B UCACCUGCUUCUUCUGGUUU 129TMPJH43A AACCAGAAGAAGCAGGUGAC 130 TMPJH43B UCACCUGCUUCUUCUGGUUC 131TMPJH44A AACCAGAAGAAGCAGGUGAG 132 TMPJH44B UCACCUGCUUCUUCUGGUUG 133TMP85A AACCAGAAGAAGCAGGUGU 134 TMP86A AACCAGAAGAAGCAGGUGC 135 TMP87AAACCAGAAGAAGCAGGUGG 136 TMP88B UCACCUGCUUCUUCUGGUA 137 TMP90BUCACCUGCUUCUUCUGGUC 138 TMP91B UCACCUGCUUCUUCUGGUG 139 ALD01AAAUGUUUUCCUGCUGACGG 140 ALD01B CCGUCAGCAGGAAAACAUU 141 ALD02AAAUGUUUUCCUGCUGACGGA 142 ALD02B CCGUCAGCAGGAAAACAUUA 143 ALD03AAAUGUUUUCCUGCUGACGGU 144 ALD03B CCGUCAGCAGGAAAACAUUU 145 ALD04AAAUGUUUUCCUGCUGACGGC 146 ALD04B CCGUCAGCAGGAAAACAUUC 147 ALD05AAAUGUUUUCCUGCUGACGGG 148 ALD05B CCGUCAGCAGGAAAACAUUG 149 ALD06AAAUGUUUUCCUGCUGACGA 150 ALD06B CCGUCAGCAGGAAAACAUA 151 ALD07AAAUGUUUUCCUGCUGACGU 152 ALD08A AAUGUUUUCCUGCUGACGC 153 ALD08BCCGUCAGCAGGAAAACAUC 154 ALD09B CCGUCAGCAGGAAAACAUG 155 STS22006L6AUCUUCUUAAACUGAGUUUC 156 STS22006L6B GAAACUCAGUUUAAGAAGA 157 STS22009L6AAUGUAGCCGAGGAUCUUCU 158 STS22009L6B AGAAGAUCCUCGGCUACAU 159 STS16001L1AUUAUAGAGCAAGAACACUGUU 160 STS16001L1B AACAGUGUUCUUGCUCUAUAA 161STS22002L6A AAUGUUUUCCUGCUGACGG 162 STS22002L6B CCGUCAGCAGGAAAACAUU 163STS22002V8L6A AAUGUUUUCCUGCUGACGA 164 STS22002V9L6B CCGUCAGCAGGAAAACAUA165 STS22009V1L6B AGAAGAUCCUCGGCUACAA 166 STS18001L4AmU(ps)fC(ps)mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA(ps)fC(ps)mG 167 STS18001L4B[ST23(ps)]3 ST41(ps)fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUmUfC(ps)mG(ps)fA 168STS16001V4L11AmU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU(ps)GlyC3Am(GaINAc)169 STS16001V4L11BfA(ps)mA(ps)fCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 170STS16001L22A mU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU171 STS16001L22BSer(GN)(ps)Ser(GN)(ps)Ser(GN)(ps)fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 172 STS16001V7L22A(po)ivAfUmAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU 173STS16001V7L22BSer(GN)(ps)Ser(GN)(ps)Ser(GN)(ps)fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 174 STS16001V8L22A(po)ivGfUmAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU 175STS16001V8L22BSer(GN)(ps)Ser(GN)(ps)Ser(GN)(ps)fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 176 STS16001V9L22A(po)ivUfUmAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU 177STS16001V9L22BSer(GN)(ps)Ser(GN)(ps)Ser(GN)(ps)fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 178 STS16001V10L22A(po)ivCfUmAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU 179STS16001V10L22BSer(GN)(ps)Ser(GN)(ps)Ser(GN)(ps)fAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA 180 STS16001V6L11AmU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU(ps)GlyC3Am(GaINAc)181 STS16001V6L11B ivAfAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA182 STS16001V7L11AmU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU(ps)GlyC3Am(GaINAc)183 STS16001V7L11B ivGfAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA184 STS16001V8L11AmU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU(ps)GlyC3Am(GaINAc)185 STS16001V8L11B ivUfAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA186 STS16001V9L11AmU(ps)fU(ps)mAfUmAfGmAfGmCfAmAfGmAfAmCfAmCfUmG(ps)fU(ps)mU(ps)GlyC3Am(GaINAc)187 STS16001V9L11B ivCfAmAfCmAfGmUfGmUfUmCfUmUfGmCfUmCfUmAfU(ps)mA(ps)fA188 STS18001L4A UCGAAGUAUUCCGCGUACG 189 STS18001L4B CGUACGCGGAAUACUUCGA190 STS16001V4L11A UUAUAGAGCAAGAACACUGUU 191 STS16001V7L22AAUAUAGAGCAAGAACACUGUU 192 STS16001V8L22A GUAUAGAGCAAGAACACUGUU 193STS16001V10L22A CUAUAGAGCAAGAACACUGUU 194 STS16001V4L11BAACAGUGUUCUUGCUCUAUAA 195 STS16001V6L11B AAACAGUGUUCUUGCUCUAUAA 196STS16001V7L11B GAACAGUGUUCUUGCUCUAUAA 197 STS16001V8L11BUAACAGUGUUCUUGCUCUAUAA 198 STS16001V9L11B CAACAGUGUUCUUGCUCUAUAA

Key

mA, mU, mC, mG—2′-OMe RNA

fA, fU, fC, fG—2′-F RNA

ivA, ivU, ivC, ivG—inverted RNA (3′-3′ from SEQ ID NO 1-122; 5′-5′ fromSEQ ID NO 166-187)

(po)ivA, (po)ivU, (po)ivC, (po)ivG: 5′-5′-linked inverted ribonucleotidewith 3′-phosphate

(ps)—phosphorothioate

The sequences listed above may be disclosed with a linker or ligand,such as GalNAC or (ps) or (ps2) linkages for example. These form anoptional, but preferred, part of the sequence of the sequence listing.

The following abbreviations may be used:

ivN Inverted nucleotide, either 3′-3′ or 5′-5′ (ps2) Phosphorodithioatevinylphosphonate Vinyl-(E)-phosphonate FAM 6-Carboxyfluorescein TAMRA5-Carboxytetramethylrhodamine BHQ1 Black Hole Quencher 1 (ps)Phosphorothioate GN

GN2

GN3

GNo Same as GN2 but with phosphodiesters instead of phosphorothioatesST23

ST41/C4XLT

ST43/C6XLT

Long trebler/ltrb/STKS

Ser(GN)

GlyC3Am(GalNAc)

GalNAc (only in when GN2 (see above) used in sequences) (MOE-U), (MOE-C)2′methoxyethyl RNA {A}, {U}, {C}, {G} LNA [ST23 (ps)]3 ST41 (ps) GN2(see above) [ST23 (ps)]3 ST43 (ps) GN3 (see above) ST23(ps) long GN (seeabove) trebler(ps)

STATEMENTS OF INVENTION

The statements reflect preferred features of the invention, and may eachindependently be combined with any aspect of the disclosure herein.

1. A nucleic acid for inhibiting expression of a target gene in a cell,comprising at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of RNAtranscribed from said target gene to be inhibited and wherein theterminal nucleotide at the 3′ end of at least one of the first strandand the second strand is an inverted nucleotide and is attached to theadjacent nucleotide via the 3′ carbon of the terminal nucleotide and the3′ carbon of the adjacent nucleotide and/or the terminal nucleotide atthe 5′ end of at least one of the first strand and the second strand isan inverted nucleotide and is attached to the adjacent nucleotide viathe 5′ carbon of the terminal nucleotide and the 5′ carbon of theadjacent nucleotide.

2. A nucleic acid according to statement 1, wherein the 3′ and/or 5′inverted nucleotide of the first and/or second strand is attached to theadjacent nucleotide via a phosphate group by way of a phosphodiesterlinkage.

3. A nucleic acid according to statement 1, wherein the 3′ and/or 5′inverted nucleotide of the first and/or second strand is attached to theadjacent nucleotide via a phosphorothioate group.

4. A nucleic acid according to statement 1, wherein 3′ and/or 5′inverted nucleotide of the first and/or second strand is attached to theadjacent nucleotide via a phosphorodithioate group.

5. A nucleic acid according to any of statements 1 to 6, wherein the 3′and/or 5′ inverted nucleotide of the first and/or second strand forms anoverhang.

6. A nucleic acid according to any of statements 1 to 5, wherein the 3′and/or 5′ inverted nucleotide of the first and/or second strand forms ablunt end.

7. A nucleic acid according to any of statements 1 to 6, wherein thefirst strand and the second strand are separate strands.

8. A nucleic acid according to any of statements 1 to 6, comprising asingle strand that comprises the first strand and the second strand.

9. A nucleic acid according to any of statements 1 to 8, wherein saidfirst strand and/or said second strand are each from 17-35 nucleotidesin length.

10. A nucleic acid of any of statements 1 to 9, wherein the at least oneduplex region consists of 19-25 nucleotide base pairs.

11. A nucleic acid of any preceding statement, which

-   -   a) is blunt ended at both ends; or    -   b) has an overhang at one end and a blunt end at the other; or    -   c) has an overhang at both ends        optionally a nucleic acid having an overhang at the 3′ end of        the first strand and which has a blunt end at the 3′ of the        second strand,        optionally wherein the nucleic acid has an inverted nucleotide        such as ivA on the 3′ end of the second strand 3′-end at a blunt        end.

12. A nucleic acid according to any preceding statement, wherein one ormore nucleotides on the first and/or second strand are modified, to formmodified nucleotides.

13. A nucleic acid of statement 12, wherein one or more of the oddnumbered nucleotides of the first strand are modified.

14. A nucleic acid according to statement 13, wherein one or more of theeven numbered nucleotides of the first strand are modified by at least asecond modification, wherein the at least second modification isdifferent from the modification of statement 9.

15. A nucleic acid of statement 14, wherein at least one of the one ormore modified even numbered nucleotides is adjacent to at least one ofthe one or more modified odd numbered nucleotides.

16. A nucleic acid of any one of statements 13 to 15, wherein aplurality of odd numbered nucleotides are modified.

17. A nucleic acid of any one of statements 14 to 16, wherein aplurality of even numbered nucleotides are modified by a secondmodification.

18. A nucleic acid of any of statements 12 to 17, wherein the firststrand comprises adjacent nucleotides that are modified by a commonmodification.

19. A nucleic acid of any of statements 13 to 18, wherein the firststrand comprises adjacent nucleotides that are modified by a secondmodification that is different to the modification of statement 9.

20. A nucleic acid of any of statements 13 to 19, wherein one or more ofthe odd numbered nucleotides of the second strand are modified by amodification that is different to the modification of statement 9.

21. A nucleic acid according to any of statements 13 to 19, wherein oneor more of the even numbered nucleotides of the second strand aremodified by the modification of statement 9.

22. A nucleic acid of statement 20 or 21, wherein at least one of theone or more modified even numbered nucleotides of the second strand isadjacent to the one or more modified odd numbered nucleotides.

23. A nucleic acid of any of statements 20 to 22, wherein a plurality ofodd numbered nucleotides of the second strand are modified by a commonmodification.

24. A nucleic acid of any of statements 20 to 23, wherein a plurality ofeven numbered nucleotides are modified by a modification according tostatement 9.

25. A nucleic acid of any of statements 20 to 24, wherein a plurality ofodd numbered nucleotides are modified by a second modification, whereinthe second modification is different from the modification of statement9.

26. A nucleic acid of any of statements 20 to 25, wherein the secondstrand comprises adjacent nucleotides that are modified by a commonmodification.

27. A nucleic acid of any of statements 20 to 26, wherein the secondstrand comprises adjacent nucleotides that are modified by a secondmodification that is different from the modification of statement 9.

28. A nucleic acid according to any one of statements 12 to 27, whereineach of the odd numbered nucleotides in the first strand and each of theeven numbered nucleotides in the second strand are modified with acommon modification.

29. A nucleic acid of any one of statements 13 to 28, wherein each ofthe even numbered nucleotides are modified in the first strand with asecond modification and each of the odd numbered nucleotides aremodified in the second strand with a second modification.

30. A nucleic acid according to any one of statements 20 to 29, whereinthe modified nucleotides of the first strand are shifted by at least onenucleotide relative to the unmodified or differently modifiednucleotides of the second strand.

31. A nucleic acid of any one of statements 1 to 30, wherein the firststrand comprises a sequence selected from the group consisting of SEQ IDNO:s 1, 3, 5 and 7.

32. A nucleic acid of any one of statements 1 to 30, wherein the secondstrand comprises a sequence selected from the group consisting to SEQ IDNO:s 2, 4, 6 and 8.

33. A nucleic acid according to any one of statements 8 to 32, whereinthe modification and/or modifications are each and individually selectedfrom the group consisting of 3′-terminal deoxy-thymine, 2′-O-methyl, a2′-deoxy-modification, a 2′-amino-modification, a 2′-alkyl-modification,a morpholino modification, a phosphoramidate modification,5′-phosphorothioate group modification, a 5′ phosphate or 5′ phosphatemimic modification and a cholesteryl derivative or a dodecanoic acidbisdecylamide group modification.

34. A nucleic acid according to any one of statements 8 to 33, whereinthe modification is any one of a locked nucleotide, an abasic nucleotideor a non-natural base comprising nucleotide.

35. A nucleic acid according to any one of statements 8 to 34, whereinat least one modification is 2′-O-methyl.

36. A nucleic acid according to any one of statements 8 to 35, whereinat least one modification is 2′-F.

37. A nucleic acid according to any one of statements 1 to 36, whereinthe inverted nucleotide at the 3′ end of at least one of the firststrand and the second strand and/or the inverted nucleotide at the 5′end of at least one of the first strand and the second strand is apurine, such as an adenine

38. A nucleic acid according to any one of statements 1 to 37, furthercomprising a ligand.

39. A nucleic acid according to any one of statements 1 to 38,comprising a phosphorothioate linkage between the terminal one, two orthree 3′ nucleotides and/or 5′ nucleotides of the first and/or thesecond strand.

40. A nucleic acid according to any one of statements 1 to 39,comprising two phosphorothioate linkage between each of the threeterminal 3′ and between each of the three terminal 5′ nucleotides on thefirst strand, and two phosphorothioate linkages between the threeterminal nucleotides of the 3′ end of the second strand.

41. A nucleic acid for inhibiting expression of a target gene in a cell,comprising at least one duplex region that comprises at least a portionof a first strand and at least a portion of a second strand that is atleast partially complementary to the first strand, wherein said firststrand is at least partially complementary to at least a portion of RNAtranscribed from said target gene to be inhibited and wherein theterminal nucleotide at the 3′ end of at least one of the first strandand the second strand is an inverted nucleotide and is attached to theadjacent nucleotide via the 3′ carbon of the terminal nucleotide and the3′ carbon of the adjacent nucleotide and/or the terminal nucleotide atthe 5′ end of at least one of the first strand and the second strand isan inverted nucleotide and is attached to the adjacent nucleotide viathe 5′ carbon of the terminal nucleotide and the 5′ carbon of theadjacent nucleotide, and wherein the nucleic acid molecule is directlyor indirectly conjugated to a ligand via a linker.

42. A nucleic acid according to any of statements 38 to 41, wherein theligand comprises one or more GalNac ligands and derivatives thereof,such as comprising a GalNAc moiety at the second strand 5′-end.

43. A nucleic acid according to any of statements 38 to 42, wherein theligand is directly or indirectly conjugated to a nucleic acid as definedin any preceding statements by a bivalent or trivalent branched linker.

44. A nucleic acid of statement 41, wherein the nucleotides are modifiedas defined in any preceding statements.

45. A nucleic acid of any preceding statement, wherein the ligandcomprises the formula I:

[S—X¹—P—X²]₃-A-X³—  (I)

wherein:

-   -   S represents a saccharide, wherein the saccharide is N-acetyl        galactosamine;    -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein        m is 1, 2, or 3;    -   P is a phosphate or modified phosphate (preferably a        thiophosphate);    -   X² is alkylene or an alkylene ether of the formula        (—CH2)_(n)—O—CH2- where n=1-6;    -   A is a branching unit;    -   X³ represents a bridging unit;    -   wherein a nucleic acid according to the present invention is        conjugated to X³ via a phosphate or modified phosphate        (preferably a thiophosphate).

46. A conjugated nucleic acid having one of the following structures:

wherein Z is a nucleic acid according to any of statements 1 to 40.

47. A nucleic acid of any preceding statement, wherein the ligandcomprises:

48. A composition comprising a nucleic acid or conjugated nucleic acidas defined in any preceding statement and a formulation comprising:

i) a cationic lipid, or a pharmaceutically acceptable salt thereof;

ii) a steroid;

iii) a phosphatidylethanolamine phospholipid;

iv) a PEGylated lipid.

49. A composition according to statement 48, wherein in the formulationthe content of the cationic lipid component is from about 55 mol % toabout 65 mol % of the overall lipid content of the lipid formulation,preferably about 59 mol % of the overall lipid content of the lipidformulation.

50. A composition as disclosed in statement 48, wherein the formulationcomprises;

A cationic lipid having the structure;

the steroid has the structure;

the phosphatidylethanolamine phospholipid has the structure;

and the PEGylated lipid has the structure;

51. A composition comprising a nucleic acid or conjugated nucleic acidof any of statements 1 to 47 and a physiologically acceptable excipient.

52. A nucleic acid or conjugated nucleic acid according to any ofstatements 1 to 47 for use in the treatment of a disease or disorder.

53. Use of a nucleic acid or conjugated nucleic acid according to any ofstatements 1 to 47 in the manufacture of a medicament for treating adisease or disorder.

54. A method of treating a disease or disorder comprising administrationof a composition comprising a nucleic acid or conjugated nucleic acidaccording to any of statements 1 to 47 to an individual in need oftreatment.

55. The method of statement 54, wherein the nucleic acid or conjugatednucleic acid is administered to the subject subcutaneously orintravenously.

56. A process of making a nucleic acid or conjugated nucleic acid of anyof statements 1 to 47.

57 A nucleic acid, method, use or composition according to any precedingstatement, or any disclosure herein, wherein there is no terminalphosphorothioate in the nucleic acid.

58 A nucleic acid, method, use or composition according to any precedingstatement, or any disclosure herein, wherein the terminal nucleotide islocated at the 3′ end of at least one of the first strand and the secondstrand, or both.

59 A nucleic acid, method, use or composition according to any precedingstatement, or any disclosure herein, wherein the ligand does not containa phosphorothioate, such as a nucleic acid according conjugated to aGalnac moiety which does not contain phosphorothioates.

1. A nucleic acid for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said target gene to be inhibited and wherein the terminal nucleotide at the 3′ end of at least one of the first strand and the second strand is an inverted nucleotide and is attached to the adjacent nucleotide via the 3′ carbon of the terminal nucleotide and the 3′ carbon of the adjacent nucleotide and/or the terminal nucleotide at the 5′ end of at least one of the first strand and the second strand is an inverted nucleotide and is attached to the adjacent nucleotide via the 5′ carbon of the terminal nucleotide and the 5′ carbon of the adjacent nucleotide.
 2. A nucleic acid of any preceding claim, which a) is blunt ended at both ends; or b) has an overhang at one end and a blunt end at the other; or c) has an overhang at both ends optionally a nucleic acid having an overhang at the 3′ end of the first strand and which has a blunt end at the 3′ of the second strand, or optionally wherein the nucleic acid has an inverted nucleotide such as ivA on the 3′ end of the second strand 3′-end at a blunt end.
 3. A nucleic acid according to any preceding claim, wherein one or more nucleotides on the first and/or second strand are modified, to form modified nucleotides.
 4. A nucleic acid according to any one of claims 1-3, wherein at least one modification is 2′-O-methyl or 2′-F.
 5. A nucleic acid according to any one of claims 1 to 4, wherein the inverted nucleotide at the 3′ end of at least one of the first strand and the second strand and/or the inverted nucleotide at the 5′ end of at least one of the first strand and the second strand is a purine, such as an adenine
 6. A nucleic acid according to any one of claims 1 to 5, further comprising a ligand.
 7. A nucleic acid for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said target gene to be inhibited and wherein the terminal nucleotide at the 3′ end of at least one of the first strand and the second strand is an inverted nucleotide and is attached to the adjacent nucleotide via the 3′ carbon of the terminal nucleotide and the 3′ carbon of the adjacent nucleotide and/or the terminal nucleotide at the 5′ end of at least one of the first strand and the second strand is an inverted nucleotide and is attached to the adjacent nucleotide via the 5′ carbon of the terminal nucleotide and the 5′ carbon of the adjacent nucleotide, and wherein the nucleic acid molecule is directly or indirectly conjugated to a ligand via a linker.
 8. A nucleic acid according to any of claim 7, wherein the ligand comprises one or more GalNac ligands and derivatives thereof, such as comprising a GalNAc moiety at the second strand 5′-end.
 9. A nucleic acid of claim 8, wherein the nucleotides are modified as defined in any preceding claims.
 10. A composition comprising a nucleic acid or conjugated nucleic acid of any of claims 1 to 9 and a physiologically acceptable excipient.
 11. A nucleic acid or conjugated nucleic acid according to any of claims 1 to 9 or composition according to claim 10 for use in the treatment of a disease or disorder.
 12. Use of a nucleic acid or conjugated nucleic acid or composition according to any of claims 1-11 in the manufacture of a medicament for treating a disease or disorder.
 13. A method of treating a disease or disorder comprising administration of a composition comprising a nucleic acid or conjugated nucleic acid according to any of claims 1 to 11 to an individual in need of treatment. 